Inducing cellular immune responses to hepatitis B virus using peptide compositions

ABSTRACT

This invention uses our knowledge of the mechanisms by which antigen is recognized by T cells to develop epitope-based vaccines directed towards HBV. More specifically, this application communicates our discovery of pharmaceutical compositions and methods of use in the prevention and treatment of HBV infection.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is divisional of U.S. application Ser. No. 09/239,043,now U.S. Pat. No. 6,689,363 filed Jan. 27, 1999, which is hereinincorporated by reference. This application is related to U.S. Ser. No.08/820,360 filed Mar. 12, 1997 and now abandoned, which claims thebenefit of U.S. Provisional Application No. 60/013,363 filed Mar. 13,1996 and now abandoned. The present application is also related to U.S.Ser. No. 09/189,702 filed Nov. 10, 1998, which is a CIP of U.S. Ser. No.08/205,713 filed Mar. 4, 1994 and now abandoned, which is a CIP of Ser.No. 08/159,184 filed Nov. 29, 1993 and now abandoned, which is a CIP of08/073,205 filed Jun. 4, 1993 and now abandoned, which is a CIP of Ser.No. 08/027,146 filed Mar. 5, 1993 and now abandoned. The presentapplication is also related to U.S. Ser. No. 08/197,484, now U.S. Pat.No. 6,419,931, abandoned U.S. Ser. No. 08/464,234, U.S. Ser. No.08/464,496, now U.S. Pat. No. 6,322,789, abandoned U.S. Ser. No.08/464,031, abandoned U.S. Ser. No. 08/464,433, and abandoned U.S. Ser.No. 08/461,603, which is a continuation of abandoned U.S. Ser. No.07/935,811, which is a CIP of abandoned U.S. Ser. No. 07/874,491, whichis a CIP of abandoned U.S. Ser. No. 07/827,682, which is a CIP ofabandoned 07/749,568. The present application is also related to U.S.patent application Ser. No. entitled “Peptides and Methods for CreatingSynthetic Peptides with Modulated Binding Affinity for HLA Molecules”,filed Jan. 6, 1999, which is a CIP of abandoned U.S. Ser. No.08/815,396, which is a CIP of abandoned U.S. Ser. No. 60/013,113.Furthermore, the present application is related to abandoned U.S. Ser.No. 09/017,735, which is a CIP of abandoned U.S. Ser. No. 08/589,108;abandoned U.S. Ser. No. 08/753,622, abandoned U.S. Ser. No. 08/822,382,abandoned U.S. Ser. No. 60/013,980, abandoned U.S. Ser. No. 08/454,033,abandoned U.S. Ser. No. 09/116,424, abandoned U.S. Ser. No. 08/205,7 13,and abandoned U.S. Ser. No. 08/349,177, which is a CIP of abandoned U.S.Ser. No. 08/159,184, which is a CIP of abandoned U.S. Ser. No.08/073,205, which is a CIP of abandoned U.S. Ser. No. 08/027,146. Thepresent application is also related to abandoned U.S. Ser. No.09/017,524, abandoned U.S. Ser. No. 08/821,739, abandoned U.S. Ser. No.60/013,833, abandoned U.S. Ser. No. 08/758,409, abandoned U.S. Ser. No.08/589,107, abandoned U.S. Ser. No. 08/451,913, U.S. Ser. No.08/186,266, now U.S. Pat. No. 5,662,907, abandoned U.S. Ser. No.09/116,061, and abandoned U.S. Ser. No. 08/347,610, which is a CIP ofU.S. Ser. No. 08/159,339, now U.S. Pat. No. 6,037,135, which is a CIP ofabandoned U.S. Ser. No. 08/103,396, which is a CIP of abandoned U.S.Ser. No. 08/027,746, which is a CIP of abandoned U.S. Ser. No.07/926,666. The present application is also related to abandoned U.S.Ser. No. 09/017,743, abandoned U.S. Ser. No. 08/753,615; U.S. Ser. No.abandoned 08/590,298, abandoned U.S. Ser. No. 09/115,400, and abandonedU.S. Ser. No. 08/452,843, which is a CIP of abandoned U.S. Ser. No.08/344,824, which is a CIP of abandoned U.S. Ser. No. 08/278,634. Thepresent application is also related to provisional U.S. Ser. No.60/087,192 and U.S. Ser. No. 09/009,953, now U.S. Pat. No. 6,413,517,which is a CIP of abandoned U.S. Ser. No. 60/036,713 and abandoned U.S.Ser. No. 60/037,432. In addition, the present application is related toabandoned U.S. Ser. No. 09/098,584 and to Provisional U.S. patentapplication entitled “Identification of Broadly Reactive HLA RestrictedT Cell Epitopes”, filed of even date herewith. All of the aboveapplications are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was funded, in part, by the United States governmentunder grants with the National Institutes of Health. The U.S. governmenthas certain rights in this invention.

INDEX

-   I. Background of the Invention-   II. Summary of the Invention-   III. Brief Description of the Figures-   IV. Detailed Description of the Invention    -   A. Definitions    -   B. Stimulation of CTL and HTL responses against HBV    -   C. Immune Response Stimulating Peptides        -   1. Binding Affinity of the Peptides for HLA Molecules        -   2. Peptide Binding Motifs and Supermotifs            -   a) HLA-A1 supermotif            -   b) HLA-A2 supermotif            -   c) HLA-A3 supermotif            -   d) HLA-A24 supermotif            -   e) HLA-B7 supermotif            -   f) HLA-B27 supermotif            -   g) HLA-B44 supermotif            -   h) HLA-B58 supermotif            -   i) HLA-B62 supermotif            -   j) HLA-A1 motif            -   k) HLA-A3 motif            -   1) HLA-A11 motif            -   m) HLA-A24 motif            -   n) HLA-A2.1 motif            -   o) HLA-DR-1-4-7 supermotif            -   p) HLA-DR3 motifs        -   3. Enhancing Population Coverage of the Vaccine    -   D. Immune Response Stimulating Peptide Analogs    -   E. Computer Screening of Protein Sequences from Disease-Related        Antigens for Supermotif or Motif Containing Peptides    -   F. Assays to Detect T-Cell Responses    -   G. Preparation of Peptides    -   H. Use of Peptide Epitopes for Evaluating Immune Responses    -   I. Vaccine Compositions        -   1. Minigene Vaccines        -   2. Combinations with Helper Peptides    -   J. Administration of Vaccines for Therapeutic or Prophylactic        Purposes    -   K. Kits-   V. Examples

I. BACKGROUND OF THE INVENTION

Chronic infection by hepatitis B virus (HBV) affects at least 5% of theworld's population and is a major cause of cirrhosis and hepatocellularcarcinoma (Hoofnagle, J., N. Engl. J. Med. 323:337, 1990; Fields, B. andKnipe, D., In: Fields Virology 2:2137, 1990). The World HealthOrganization lists hepatitis B as a leading cause of death worldwide,close behind chronic pulmonary disease, and more prevalent than AIDS.Chronic HBV infection can range from an asymptomatic carrier state tocontinuous hepatocellular necrosis and inflammation, and can lead tohepatocellular carcinoma.

The immune response to HBV is believed to play an important role incontrolling hepatitis B infection. A variety of humoral and cellularresponses to different regions of the HBV nucleocapsid core and surfaceantigens have been identified. T cell mediated immunity, particularlyinvolving class I human leukocyte antigen-restricted cytotoxic Tlymphocytes (CTL), is believed to be crucial in combatting establishedHBV infection.

Class I human leukocyte antigen (HLA) molecules are expressed on thesurface of almost all nucleated cells. CTL recognize peptide fragments,derived from intracellular processing of various antigens, in the formof a complex with class I HLA molecules. This recognition event thenresults in the destruction of the cell bearing the HLA-peptide complexdirectly or the activation of non-destructive mechanisms e.g., theproduction of interferon, that inhibit viral replication.

Several studies have emphasized the association between self-limitingacute hepatitis and multispecific CTL responses (Penna, A. et al., J.Exp. Med. 174:1565, 1991; Nayersina, R. et al., J. Immunol. 150:4659,1993). Spontaneous and interferon-related clearance of chronic HBVinfection is also associated with the resurgence of a vigorous CTLresponse (Guidotti, L. G. et al., Proc. Natl. Acad. Sci. USA 91:3764,1994). In all such cases the CTL responses are polyclonal, and specificfor multiple viral proteins including the HBV envelope, core andpolymerase antigens. By contrast, in patients with chronic hepatitis,the CTL activity is usually absent or weak, and antigenicallyrestricted.

The crucial role of CTL in resolution of HBV infection has been furtherunderscored by studies using HBV transgenic mice. Adoptive transfer ofHBV-specific CTL into mice transgenic for the HBV genome resulted insuppression of virus replication. This effect was primarily mediated bya non-lytic, lymphokine-based mechanism (Guidotti, L. G. et al., Proc.Natl. Acad. Sci. USA 91:3764, 1994; Guidotti, L. G., Guilhot, S., andChisari, F. V. J. Virol. 68:1265, 1994; Guidotti, L. G. et al., J.Virol. 69:6158, 1995; Gilles, P. N., Fey, G., and Chisari, F. V., J.Virol. 66:3955, 1992).

As is the case for HLA class I restricted responses, HLA class IIrestricted T cell responses are usually detected in patients with acutehepatitis, and are absent or weak in patients with chronic infection(Chisari, F. V. and Ferrari, C., Annu. Rev. Immunol. 1′:29, 1995). HLAClass II responses are tied to activation of helper T cells (IT Ls)Helper T lymphocytes, which recognize Class II HLA molecules, maydirectly contribute to the clearance of HBV infection through thesecretion of cytokines which suppress viral replication (Franco, A. etal., J. Immunol. 159:2001, 1997). However, their primary role in diseaseresolution is believed to be mediated by inducing activation andexpansion of virus-specific CTL and B cells.

In view of the heterogeneous immune response observed with HBVinfection, induction of a multi-specific cellular immune responsedirected simultaneously against multiple epitopes appears to beimportant for the development of an efficacious vaccine against HBV.There is a need to establish vaccine embodiments that elicit immuneresponses that correspond to responses seen in patients that clear HBVinfection. Epitope-based vaccines appear useful.

Upon development of appropriate technology, the use of epitope-basedvaccines has several advantages over current vaccines. The epitopes forinclusion in such a vaccine are to be selected from conserved regions ofviral or tumor-associated antigens, in order to reduce the likelihood ofescape mutants. The advantage of an epitope-based approach over the useof whole antigens is that there is evidence that the immune response towhole antigens is directed largely toward variable regions of theantigen, allowing for immune escape due to mutations. Furthermore,immunosuppressive epitopes that may be present in whole antigens can beavoided with the use of epitope-based vaccines.

Additionally, with an epitope-based vaccine approach, there is anability to combine selected epitopes (CTL and HTL) and additionally tomodify the composition of the epitopes, achieving, for example, enhancedimmunogenicity. Accordingly, the immune response can be modulated, asappropriate, for the target disease. Similar engineering of the responseis not possible with traditional approaches.

Another major benefit of epitope-based immune-stimulating vaccines istheir safety. The possible pathological side effects caused byinfectious agents or whole protein antigens, which might have their ownintrinsic biological activity, is eliminated.

An epitope-based vaccine also provides the ability to direct and focusan immune response to multiple selected antigens from the same pathogen.Thus, patient-by-patient variability in the immune response to aparticular pathogen may be alleviated by inclusion of epitopes frommultiple antigens from that pathogen in a vaccine composition. A“pathogen” may be an infectious agent or a tumor associated molecule.

However, one of the most formidable obstacles to the development ofbroadly efficacious epitope-based immunotherapeutics has been theextreme polymorphism of HLA molecules. To date, effectivenon-genetically biased coverage of a population has been a task ofconsiderable complexity; such coverage has required that epitopes beused specific for HLA molecules corresponding to each individual HLAallele, therefore, impractically large numbers of epitopes would have tobe used in order to cover ethnically diverse populations. There hasexisted a need to develop peptide epitopes that are bound by multipleHLA antigen molecules for use in epitope-based vaccines. The greater thenumber of HLA antigen molecules bound, the greater the breadth ofpopulation coverage by the vaccine.

Furthermore, as described herein in greater detail, a need has existedto modulate peptide binding properties, for example so that peptidesthat are able to bind to multiple HLA antigens do so with an affinitythat will stimulate an immune response. Identification of epitopesrestricted by more than one HLA allele at an affinity that correlateswith immunogenicity is important to provide thorough populationcoverage, and to allow the elicitation of responses of sufficient vigorwhereby the natural immune responses noted in self-limiting acutehepatitis, or of spontaneous clearance of chronic HBV infection isinduced in a diverse segment of the population. Such a response can alsotarget a broad array of epitopes. The technology disclosed hereinprovides for such favored immune responses.

The information provided in this section is intended to disclose thepresently understood state of the art as of the filing date of thepresent application. Information is included in this section which wasgenerated subsequent to the priority date of this application.Accordingly, background in this section is not intended, in any way, todelineate the priority date for the invention.

II. SUMMARY OF THE INVENTION

This invention applies our knowledge of the mechanisms by which antigenis recognized by T cells, for example, to develop epitope-based vaccinesdirected towards HBV. More specifically, this application communicatesour discovery of specific epitope pharmaceutical compositions andmethods of use in the prevention and treatment of HBV infection.

An embodiment of the present invention includes a peptide composition ofless than 100 amino acid residues comprising a peptide epitope usefulfor inducing an immune response against hepatitis B virus (HBV) saidepitope (a) having an amino acid sequence of about 8 to about 13 aminoacid residues that have at least 65% identity with a native amino acidsequence for HBV, and, (b) binding to at least one MHC class I HLAallele with a dissociation constant of less than about 500 nM. Further,the peptide composition may comprise an amino acid sequence of at least77% identity, or at least 100% identity with a native HBV amino acidsequence. In a preferred embodiment, the peptide is one of the peptidesdesignated as being from the envelope, polymerase, protein X, ornucleocapsid core regions of HBV. Preferred peptides are described inTables VI through XVII or XXI.

An additional embodiment of the present invention comprises acomposition of less than 100 amino acid residues comprising a peptideepitope useful for inducing an immune response against hepatitis B virus(HBV) said peptide (a) having an amino acid sequence of about 8 to about13 amino acid residues and (b) bearing one of the HLA supernotifs ormotifs set out in Tables I and II. Furthermore, the composition maycomprise a peptide wherein the peptide is one of those described inTables VI through XVII or Table XXI which bear an HLA A1, A2, A3, A24,B7, B27, B44, B58, or B62 supermotif; or an HLA A1, A3, A11, A24, orA2.1 motif or an HLA A*3301, A*3101, A*6801, B*0702, B*3501, B51,B*5301, B*5401 motif.

In one embodiment of a peptide comprising an HLA A2.1 motif, the peptidedoes not bear an L or M at position 2 and V at the C-terminal position 9of a 9 amino acid peptide.

An alternative embodiment of the invention comprises an analog of an HBVpeptide of less than 100 amino acid residues in length that bears an HLAbinding motif, the analog bearing the same HLA binding motif as thepeptide but comprising at least one anchor residue that is differentfrom that of the peptide. In a preferred embodiment, said peptide is ananalog of a peptide described in Table VI through Table XVII bearing anHLA A1, A2, A3, A24, B7, B27, B44, B58, or B62 supermotif; or an HLA A1,A3, A11, A24, or A2.1 motif or A3301, A3101, A6801, B0702, B3501, B51,B5301, B5401 motif.

Embodiments of the invention further include a composition of less than100 amino acid residues comprising a peptide epitope useful for inducingan immune response against hepatitis B virus (HBV) said peptide (a)having an amino acid sequence of about 9 to about 25 amino acid residuesthat have at least 65% identity with a native amino acid sequence forHBV and (b) binding to at least one MHC class II HLA allele with adissociation constant of less than about 1000 nM. In a preferredembodiment, the composition comprises a peptide that has at least 77%,or, 100% identity with a native HBV amino acid sequence. Further, thecomposition may comprise a peptide wherein said peptide is one of thosepeptides described in Table XVIII or Table XIX.

The invention also includes a peptide composition of less than 100 aminoacid residues, said composition comprising an epitope useful forinducing an immune response against hepatitis B virus (HBV) said epitope(a) having an amino acid sequence of about 10 to about 20 amino acidresidues and (b) bearing one of the class II HLA motifs set out in TableIII. In a preferred embodiment, said peptide is one of those peptidesdescribed in Table XVIII or XIX.

Additional embodiments of the invention include a composition thatcomprises an isolated nucleic acid sequence that encodes one of thepeptides set out in Tables VI through XIX or XXI or XXIII.

Alternatively, an embodiment of the invention comprises a compositionthat comprises at least two peptides, at least one of said at least twopeptides selected from Tables VI-XIX or XXI or XXIII. In a preferredembodiment, two or more of the at least two peptides are depicted inTables VI-XIX or XXI or XXIII. The composition may further comprise atleast one nucleic acid sequence. In a preferred embodiment each of saidat least two peptides are encoded by a nucleic acid sequence, whereineach of the nucleic acid sequences are located on a single vector.

Embodiments of the invention additionally include a peptide compositionof less than 100 amino acid residues, said composition comprising anepitope useful for inducing an immune response against HBV, said epitopehaving at least one of the amino acid sequences set out in Table XXIII.

An alternative modality for defining the peptides in accordance with theinvention is to recite the physical properties, such as length; primary,secondary and/or tertiary structure; or charge, which are correlatedwith binding to a particular allele-specific HLA molecule or group ofallele-specific HLA molecules. A further modality for defining peptidesis to recite the physical properties of an HLA binding pocket, orproperties shared by several allele-specific HLA binding pockets (e.g.pocket configuration and charge distribution) and reciting that thepeptide fits and binds to said pocket or pockets.

An additional embodiment of the invention comprises a method forinducing a cytotoxic T cell response to HBV in a mammal comprisingadministering to said mammal at least one peptide from Tables VI to XIXor Table XXI.

Further embodiments of the invention include a vaccine for treating HBVinfection that induces a protective immune response, wherein saidvaccine comprises at least one peptide selected from Tables VI to TableXIX or Table XXI in a pharmaceutically acceptable carrier.

Also included as an embodiment of the invention is a vaccine forpreventing HBV infection that induces a protective immune response,wherein said vaccine comprises at least one peptide selected from TablesVI to XIX or Table XXI in a pharmaceutically acceptable carrier.

The invention further includes an embodiment comprising a method forinducing a cytotoxic T cell response to HBV in a mammal, comprisingadministering to said mammal a nucleic acid sequence encoding a peptideselected from Tables VI to XIX or Table XXI.

A further embodiment of the invention comprises a kit for a vaccine fortreating or preventing HBV infection, wherein the vaccine induces aprotective immune response, said vaccine comprising at least one peptideselected from Tables VI to XIX or Table XXI in a pharmaceuticallyacceptable carrier and instructions for administration to a patient.

Lastly, the invention includes an embodiment comprising a method formonitoring immunogenic activity of a vaccine for HBV in a patient havinga known HLA-type, the method comprising incubating a T lymphocyte samplefrom the patient with a peptide selected from Tables VI to XIX or TableXXI which binds the product of at least one HLA allele present in saidpatient, and detecting for the presence of a T lymphocyte that binds tothe peptide. In a preferred embodiment, the peptide comprises atetrameric complex.

As will be apparent from the discussion below, other methods andembodiments are also contemplated. Further, novel synthetic peptidesproduced by any of the methods described herein are also part of theinvention.

III. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: FIG. 1 Illustrates the Position of Peptide Epitopes inExperimental Model Minigene Constructs

IV. DETAILED DESCRIPTION OF THE INVENTION

The peptides and corresponding nucleic acid compositions of the presentinvention are useful for stimulating an immune response to HBV either bystimulating the production of CTL or HTL responses. The peptides, whichare derived directly or indirectly from native HBV amino acid sequences,are able to bind to HLA molecules and stimulate an immune response toHBV. The complete polyprotein sequence from HBV and its variants can beobtained from Genbank. Peptides can also be readily determined fromsequence information that may subsequently be discovered for heretoforeunknown variants of HBV as will be clear from the disclosure providedbelow.

The peptides of the invention have been identified in a number of ways,as will be discussed below. Further, analog peptides have been derivedand the binding activity for HLA molecules modulated by modifyingspecific amino acid residues to create peptide analogs exhibitingaltered immunogenicity. Further, the present invention providescompositions and combinations of compositions that enable epitope-basedvaccines that are capable of interacting with multiple HLA antigens toprovide broader population coverage than prior vaccines.

The invention can be better understood with reference to the followingdefinitions:

IV.A. Definitions

“Cross-reactive binding” indicates that a peptide is bound by more thanone HLA molecule; a synonym is degenerate binding.

A “cryptic epitope” elicits a response by immunization with an isolatedpeptide, but the response is not cross-reactive in vitro when intactwhole protein which comprises the epitope is used as an antigen.

A “dominant epitope” is an epitope that induces an immune response uponimmunization with a whole native antigen. (See, e.g., Sercarz, et al.,Annu. Rev. Immunol. 11:729766 (1993)) Such a response is cross-reactivein vitro with an isolated peptide epitope.

With regard to a particular amino acid sequence, an “epitope” is a setof amino acid residues which is involved in recognition by a particularimmunoglobulin, or in the context of T cells, those residues necessaryfor recognition by T cell receptor proteins and/or MajorHistocompatibility Complex (MHC) receptors. In an immune system setting,in vivo or in vitro, an epitope is the collective features of amolecule, such as primary, secondary and tertiary peptide structure, andcharge, that together form a site recognized by an immunoglobulin, Tcell receptor or HLA molecule.

As used herein, “high affinity” with respect to HLA class I molecules isdefined as binding with an IC₅₀ (or K_(D)) of less than 50 nM.“Intermediate affinity” is binding with an IC₅₀ (or K_(D)) of betweenabout 50 and about 500 nM. “High affinity” with respect to binding toHLA class II molecules is defined as binding with an K_(D) of less than100 nM. “Intermediate affinity” is binding with a K_(D) of between about100 and about 1000 nM. Assays for determining binding are described indetail in PCT publications WO 94/20127 and WO 94/03205. Alternatively,binding is expressed relative to a reference peptide. As a particularassay becomes more, or less, sensitive, the IC₅₀'s of the peptidestested may change somewhat. However, the binding relative to thereference peptide will not significantly change. For example, in anassay run under conditions such that the IC₅₀ of the reference peptideincreases 10-fold, the IC₅₀ values of the test peptides will also shiftapproximately 10-fold. Therefore, to avoid ambiguities, the assessmentof whether a peptide is a good, intermediate, weak, or negative binderis generally based on its IC₅₀, relative to the IC₅₀ of a standardpeptide.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

An “HLA supertype or family”, as used herein, describes sets of HLAmolecules grouped on the basis of shared peptide-binding specificities.HLA class I molecules that share somewhat similar binding affinity forpeptides bearing certain amino acid motifs We grouped into HLAsupertypes. The terms HLA superfamily, HLA supertype family, and HLAxx-like supertype molecules (where xx denotes a particular HLA type) aresynonyms.

Throughout this disclosure, results are expressed in terms of “IC₅₀'s.”IC₅₀ is the concentration of peptide in a binding assay at which 50%inhibition of binding of a reference peptide is observed. Given theconditions in which the assays are run (i.e., limiting HLA proteins andlabeled peptide concentrations), these values approximate K_(D) values.It should be noted that IC₅₀ values can change, often dramatically, ifthe assay conditions are varied, and depending on the particularreagents used (e.g., HLA preparation, etc.). For example, excessiveconcentrations of HLA molecules will increase the apparent measured IC₅₀of a given ligand.

The terms “identical” or percent “identity,” in the context of two ormore peptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesthat are the same, when compared and aligned for maximum correspondenceover a comparison window, as measured using a sequence comparisonalgorithms or by manual alignment and visual inspection.

An “immunogenic peptide” or “peptide epitope” is a peptide whichcomprises an allele-specific motif or supermotif such that the peptidewill bind an HLA molecule and induce a CTL and/or HTL response. Thus,immunogenic peptides of the invention are capable of binding to anappropriate HLA molecule and thereafter inducing a cytotoxic T cellresponse, or a helper T cell response, to the antigen from which theimmunogenic peptide is derived.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment.

“Major Histocompatibility Complex” or “MHC” is a cluster of genes thatplays a role in control of the cellular interactions responsible forphysiologic immune responses. In humans, the MHC complex is also knownas the HLA complex. For a detailed description of the MHC and HLAcomplexes, see, Paul, FUNDAMENTAL IMMUNOLOGY, 3^(RD) ED., Raven Press,N.Y., 1993.

The term “motif” refers to the pattern of residues in a peptide ofdefined length, usually a peptide of from about 8 to about 13 aminoacids for a class I HLA motif and from about 6 to about 25 amino acidsfor a class II HLA motif, which is recognized by a particular HLAmolecule. Peptide motifs are typically different for each proteinencoded by each human HLA allele and differ in the pattern of theprimary and secondary anchor residues.

A “negative binding residue” is an amino acid which if present atcertain positions (typically not primary anchor positions) of peptideepitope results in decreased binding affinity of the peptide for thepeptide's corresponding HLA molecule.

The term “peptide” is used interchangeably with “oligopeptide” in thepresent specification to designate a series of residues, typicallyL-amino acids, connected one to the other, typically by peptide bondsbetween the a-amino and carboxyl groups of adjacent amino acids. Thepreferred CTL-inducing oligopeptides of the invention are fewer than 25residues in length, or less than 15 residues in length or 13 residues orless in length and usually consist of between about 8 and about 11residues, preferably 9 or 10 residues. The preferred HTL-inducingoligopeptides are less than about 50 residues in length and usuallyconsist of between about 6 and about 30 residues, more usually betweenabout 12 and 25, and often between about 15 and 20 residues.

“Pharmaceutically acceptable” refers to a non-toxic, inert, andphysiologically compatible composition.

A “primary anchor residue” is an amino acid at a specific position alonga peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding grooves ofan HLA molecule, with their side chains buried in specific pockets ofthe binding grooves themselves. In one embodiment, the primary anchorresidues are located at position 2 (from the amino terminal position)and at the carboxyl terminal position of a 9 residue peptide inaccordance with the invention. The primary anchor positions for eachmotif and supermotif are set forth in Table I. For example, analogpeptides can be created by altering the presence or absence ofparticular residues in these primary anchor positions. Such analogs areused to finely modulate the binding affinity of a peptide comprising aparticular motif or supermotif.

“Promiscuous binding” is where a distinct peptide is recognized by thesame T cell clone in the context of various HLA molecules.

A “protective immune response” refers to a CTL and/or an HTL response toan antigen from an infectious agent or a tumor antigen from which animmunogenic peptide is derived, and thereby preventing or at leastpartially arresting disease symptoms or progression. The immune responsemay also include an antibody response which has been facilitated by thestimulation of helper T cells.

The term “residue” refers to an amino acid or amino acid mimeticincorporated into an oligopeptide by an amide bond or amide bondmimetic.

A “secondary anchor residue” is an amino acid at a position other than aprimary anchor position in a peptide which may influence peptidebinding. A secondary anchor residue occurs at a significantly higherfrequency amongst bound peptides than would be expected by randomdistribution of amino acids at one position. The secondary anchorresidues are said to occur at “secondary anchor positions.” A secondaryanchor residue can be identified as a residue which is present at ahigher frequency among high affinity binding peptides, or a residueotherwise associated with high affinity binding. For example, analogpeptides can be created by altering the presence or absence ofparticular residues in these secondary anchor positions. Such analogsare used to finely modulate the binding affinity of a peptide comprisinga particular motif or supermotif.

A “subdominant epitope” is an epitope which evokes little or no responseupon immunization with whole antigens which comprise the epitope, butfor which a response can be obtained by immunization with an isolatedpeptide, and this response (unlike the case of cryptic epitopes) isdetected when whole protein is used to recall the response in vitro orin vivo.

A “supermotif” is a peptide binding specificity shared by HLA moleculesencoded by two or more HLA alleles. Thus, a preferably is recognizedwith high or intermediate affinity (as defined herein) by two or moreHLA antigens.

“Synthetic peptide” refers to a peptide that is not naturally occurring,but is man-made using such methods as chemical synthesis or recombinantDNA technology.

The nomenclature used to describe peptide compounds follows theconventional practice wherein the amino group is presented to the left(the N-terminus) and the carboxyl group to the right (the C-terminus) ofeach amino acid residue. When amino acid residue positions are referredto in a peptide epitope they are numbered in an amino to carboxyldirection with position one being the position closest to the aminoterminal. In the formulae representing selected specific embodiments ofthe present invention, the amino- and carboxyl-terminal groups, althoughnot specifically shown, are in the form they would assume at physiologicpH values, unless otherwise specified. In the amino acid structureformulae, each residue is generally represented by standard three letteror single letter designations. The L-form of an amino acid residue isrepresented by a capital single letter or a capital first letter of athree-letter symbol, and the D-form for those amino acids having D-formsis represented by a lower case single letter or a lower case threeletter symbol. Glycine has no asymmetric carbon atom and is simplyreferred to as “Gly” or G. Symbols for the amino acids are shown below.

Single Letter Symbol Three Letter Symbol Amino Acids A Ala Alanine C CysCysteine D Asp Aspartic Acid E Glu Glutamic Acid F Phe Phenylalanine GGly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu LeucineM Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R ArgArginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan YTyr TyrosineIV.B. Stimulation of CTL and HTL Responses Against HBV

The mechanism by which T cells recognize antigens has been delineatedduring the past ten years. Based on our new understanding of the immunesystem we have generated efficacious peptide epitope vaccinecompositions that can induce a therapeutic or prophylactic immuneresponse to HBV infection in a broad population. For an understanding ofthe value and efficacy of the claimed compositions, a brief review ofthe technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A., andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are described here and set forth in Tables I, II, and III(see also, e.g., Sette, A. and Grey, H. M, Curr. Opin. Immunol. 4:79,1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994; Engelhard,V. H., Curr. Opin. Immunol. 6:13, 1994). Furthermore, x-raycrystallographic analysis of HLA-peptide complexes has revealed pocketswithin the peptide binding cleft of HLA molecules which accommodateallele-specific residues borne by peptide ligands; these residues inturn determine the HLA binding capacity of the peptides in which theyare present (Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. etal., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M.et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992;Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P.J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991).

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs or class I supermotifs allows identification of regionswithin a protein that have the potential of binding particular HLAantigens (see also e.g., Sette, A. and Grey, H. M., Curr. Opin. Immunol.4:79, 1992; Sinigaglia, F. and Hammer, J., Curr. Biol. 6:52, 1994;Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994Kast, W. M. et al., J.Immunol., 152:3904, 1994).

Furthermore, a variety of assays to detect and quantify the affinity ofinteraction between peptide and HLA have also been established (Sette,A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. andHammer, J., Curr. Biol. 6:52, 1994; Engelhard, V. H., Curr. Opin.Immunol. 6:13, 1994).

We have found that the correlation of binding affinity withimmunogenicity is an important factor to be considered when evaluatingcandidate peptides. Thus, by a combination of motif searches andHLA-peptide binding assays, candidates for epitope-based vaccines havebeen identified. After determining their binding affinity, additionalconfirmatory work can be performed to select, amongst these vaccinecandidates, epitopes with desired characteristics in terms ofantigenicity and immunogenicity. Various strategies can be utilized toevaluate immunogenicity, including:

1) Primary T cell cultures from normal individuals (Wentworth, P. A. etal., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad.Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997;Kawashima, I. et al., Human Immunol. 59:1, 1998); This procedureinvolves the stimulation of PBL from normal subjects with a test peptidein the presence of antigen presenting cells in vitro over a period ofseveral weeks. T cells specific for the peptide become activated duringthis time and are detected using a ⁵¹Cr-release assay involving peptidesensitized target cells.

2) Immunization of HLA transgenic mice (Wentworth, P. A. et al., J.Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651,1996; Alexander, J. et al., J. Immunol. 159:4753, 1997); In this method,peptides in incomplete Freund's adjuvant are administered subcutaneouslyto HLA transgenic mice. Several weeks following immunization,splenocytes are removed and cultured in vitro in the presence of testpeptide for approximately one week. Peptide-specific T cells aredetected using a ⁵¹Cr-release assay involving peptide sensitized targetcells and target cells expressing endogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave recovered from infection, and/or from chronically infected patients(Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. etal., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503,1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H.M. et al., J. Virol. 71:6011, 1997). In applying this strategy, recallresponses were detected by culturing PBL from subjects that had beennaturally exposed to the antigen, for instance through infection, andthus had generated an immune response “naturally”. PBL from subjectswere cultured in vitro for 1-2 weeks in the presence of test peptideplus antigen presenting cells (APC) to allow activation of “memory” Tcells, as compared to “naive” Tcells. At the end of the culture period,T cell activity is detected using assays for T cell activity including⁵¹Cr release involving peptide-sensitized targets, T cell proliferationor lymphokine release.

The following describes the peptide epitopes and corresponding nucleicacids of the invention.

IV.C. Immune Response Stimulating Peptides

As indicated herein, the large degree of HLA polymorphism is animportant factor to be taken into account with the epitope-basedapproach to vaccine development. To address this factor, epitopeselection encompassing identification of peptides capable of binding athigh or intermediate affinity to multiple HLA molecules is preferablyutilized, most preferably these epitopes bind at high or intermediateaffinity to two or more allele specific HLA molecules.

IV.C.1. Binding Affinity of the Peptides for HLA Molecules

CTL-inducing peptides of interest for vaccine compositions preferablyinclude those that have a binding affinity for class I HLA molecules ofless than 500 nM. HTL-inducing peptides preferably include those thathave a binding affinity for class II HLA molecules of less than 1000 nM.For example, peptide binding is assessed by testing the capacity of acandidate peptide to bind to a purified HLA molecule in vitro. Peptidesexhibiting high or intermediate affinity are then considered for furtheranalysis. Selected peptides are tested on other members of the supertypefamily. In preferred embodiments, peptides that exhibit cross-reactivebinding preferably are then used in cellular screening analyses. Apeptide is considered to be an epitope if it possesses the molecularfeatures that form the binding site for a particular immunoglobulin or Tcell receptor protein.

As disclosed herein, high HLA binding affinity is correlated withgreater immunogenicity. Greater immunogenicity can be manifested inseveral different ways. Immunogenicity corresponds to whether an immuneresponse is elicited at all, and to the vigor of any particularresponse. For example, a peptide might elicit an immune response in adiverse array of the population, yet in no instance produce a vigorousresponse. In accordance with these principles, close to 90% of highbinding peptides have been found to be immunogenic, as contrasted withabout 50% of the peptides which bind with intermediate affinity.Moreover, higher binding affinity peptides leads to more vigorousimmunogenic responses. As a result, less peptide is required to elicit asimilar biological effect if a high affinity binding peptide is used.Thus, in preferred embodiments of the invention, high binding epitopesare particularly desired.

The relationship between binding affinity for HLA class I molecules andimmunogenicity of discrete peptide epitopes on bound antigens has beendetermined for the first time in the art by the present inventors. Thecorrelation between binding affinity and immunogenicity was analyzed intwo different experimental approaches (Sette, et al., J. Immunol.153:5586-5592, 1994). In the first approach, the immunogenicity ofpotential epitopes ranging in HLA binding affinity over a 10,000-foldrange was analyzed in HLA-A*0201 transgenic mice. In the secondapproach, the antigenicity of approximately 100 different hepatitis Bvirus (HBV)-derived potential epitopes, all carrying A*0201 bindingmotifs, was assessed by using PBL (peripheral blood lymphocytes) ofacute hepatitis patients. Pursuant to these approaches, it wasdetermined that an affinity threshold of approximately 500 nM(preferably 500 nM or less) determines the capacity of a peptide epitopeto elicit a CTL response. These data are true for class I bindingaffinity measurements for naturally processed peptides and forsynthesized T cell epitopes. These data also indicate the important roleof determinant selection in the shaping of T cell responses.

An affinity threshold associated with immunogenicity in the context ofHLA class II DR molecules has also been delineated (Southwood et al. J.Immunology 160:3363-3373,1998, and U.S. Ser. No. 60/087,192 filed May29, 1998). In order to define a biologically significant threshold of DRbinding affinity, a database of the binding affinities of 32DR-restricted epitopes for their restricting element was compiled. Inapproximately half of the cases (15 of 32 epitopes), DR restriction wasassociated with high binding affinities, i.e. binding affinities of lessthan 100 nM. In the other half of the cases (16 of 32), DR restrictionwas associated with intermediate affinity (binding affinities in the100-1000 nM range). In only one of 32 cases was DR restrictionassociated with an IC₅₀ of 1000 nM or greater. Thus, 1000 nM can bedefined as an affinity threshold associated with immunogenicity in thecontext of DR molecules.

The binding affinity of peptides for HLA molecules can be determined asdescribed in Example 1, below.

IV.C.2. Peptide Binding Motifs and Supermotifs

In the past few years evidence has accumulated to demonstrate that alarge fraction of HLA class I, and possibly class II molecules can beclassified into a relatively few supertypes characterized by largelyoverlapping peptide binding repertoires, and consensus structures of themain peptide binding pockets. Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues required forallele-specific binding to HLA molecules have been identified. Thesemotifs are relevant since they indicate peptides that have bindingaffinity for HLA molecules.

For HLA molecule pocket analyses, the residues comprising the B and Fpockets of HLA class I molecules as described in crystallographicstudies (Guo, H. C. et al., Nature 360:364, 1992; Saper, M. A.,Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991; Madden,D. R., Garboczi, D. N. and Wiley, D. C., Cell 75:693, 1993), have beencompiled from the database of Parham, et al. (Parham, P., Adams, E. J.,and Arnett, K. L., Immunol. Rev. 143:141, 1995). In these analyses,residues 9, 45, 63, 66, 67, 70, and 99 were considered to make up the Bpocket, and to determine the specificity for the residue in the secondposition of peptide ligands. Similarly, residues 77, 80, 81, and 116were considered to determine the specificity of the F pocket, and todetermine the specificity for the C-terminal residue of a peptide ligandbound by the HLA molecule.

Peptides of the present invention may also include epitopes that bind toMHC class II DR molecules. A significant difference between class I andclass II HLA molecules is that, although a stringent size restrictionexists for peptide binding to class I molecules, a greater degree ofheterogeneity in both sizes and binding frame positions of the motif,relative to the N and C termini of the peptide, can be demonstrated forclass II peptide ligands. This increased heterogeneity is due to thestructure of the class II-binding groove which, unlike its class Icounterpart, is open at both ends. Crystallographic analysis ofDRB*0101-peptide complexes (see, e.g., Madden, D. R. Ann. Rev. Immunol.13:587 (1995)) showed that the residues occupying position 1 andposition 6 of peptides complexed with DRB*0101 engage two complementarypockets on the DRBa*0101 molecules, with the P1 position correspondingto the most crucial anchor residue and the deepest hydrophobic pocket.Other studies have also pointed to the P6 position as a crucial anchorresidue for binding to various other DR molecules.

Thus, peptides of the present invention are identified by any one ofseveral HLA-specific amino acid motifs. If the presence of the motifcorresponds to the ability to bind several allele-specific LLA antigensit is referred to as a supermotif. The allele-specific HLA moleculesthat bind to peptides that possess a particular amino acid supermotifare collectively referred to as an HLA “supertype.”

The peptide motifs and supermotifs described below provide guidance forthe identification and use of peptides in accordance with the invention.Examples of peptide epitopes bearing the respective supermotif or motifare included in Tables as designated in the description of each motif orsupermotif. The Tables include a binding affinity ratio listing for someof the peptide epitopes. The ratio may be converted to IC₅₀ by using thefollowing formula: IC₅₀ of the standard peptide/ratio=IC₅₀ of the testpeptide (i.e. the peptide epitope). The IC₅₀ values of standard peptidesused to determine binding affinities for Class I peptides are shown inTable IV. The IC₅₀ values of standard peptides used to determine bindingaffinities for Class II peptides are shown in Table V. The peptides usedas standards for the binding assay are examples of standards;alternative standard peptides can also be used when performing such ananalysis.

To obtain the peptide epitope sequences listed in each Table, proteinsequence data from twenty HBV strains (HPBADR, HPBADR1CG, HPBADRA,HPBADRC, HPBADRCG, HPBCGADR, HPBVADRM, HPBADW, HPBADW1, HPBADW2,HPBADW3, HPBADWZ, HPBHEPB, HPBVADW2, HPBAYR, HPBV, HPBVAYWC, HPBVAYWCI,NAD HPBVAYWE) were evaluated for the presence of the designatedsupermotif or motif. Peptide epitopes were also selected on the basis oftheir conservancy. A criterion for conservancy requires that the entiresequence of a peptide be totally conserved in 75% of the sequencesavailable for a specific protein. The percent conservancy of theselected peptide epitopes is indicated on the Tables. The frequency,i.e. the number of strains of the 20 strains in which the peptidesequence was identified, is also shown. The “1^(st) position” column inthe Tables designates the amino acid position of the HBV polyproteinthat corresponds to the first amino acid residue of the epitope.Preferred peptides are designated by an asterisk.

HLA Class I Motifs Indicative of CTL Inducing Peptide Epitopes:

IV.C.2.a) HLA-A1 Supermotif

The HLA-A1 supermotif is characterized by peptides having a generalmotif of small (T or S) and hydrophobic (L, I, V, M, or F) primaryanchor residues in position 2, and aromatic (Y, F, or W) primary anchorresidues at the C-terminal position The corresponding family of HLAmolecules that bind to the A1 supermotif (the HLA-A1 supertype) includesA*0101, A*2601, A*2602, A*2501, and A*3201. (DiBrino, M. et al., J.Immunol. 151:5930, 1993; DiBrino, M. et al., J. Immunol. 152:620, 1994;Kondo, A. et al., Immunogenetics 45:249, 1997; Dumrese et al.,submitted). Peptides binding to each of the individual HLA proteins canbe modulated by substitutions at primary anchor positions.

Representative peptide epitopes that contain the A1 supermotif are setforth on the attached Table VI.

IV.C.2.b) HLA-A2 Supermotif

The HLA-A2 supermotif is characterized by the presence in peptideligands of small or aliphatic amino acids (L, I, V, M, A, T, or Q) atposition 2 and L, I, V, M, A, or T at the C-terminal position. Thesepositions ate referred to as primary anchors. The corresponding familyof HLA molecules (the HLA-A2 supertype that binds these peptides) iscomprised of at least nine HLA-A proteins: A*0201, A*0202, A*0203,A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. As explained indetail below, binding to each of the individual allele-specific HLAmolecules can be modulated by substitutions at the primary anchor and/orsecondary anchor positions.

Representative peptide epitopes that contain the A2 supermotif are setforth on the attached Table VII.

IV.C.2.c) HLA-A3 Supermotif

The HLA-A3 supermotif is characterized by peptide ligands having primaryanchor residues: A, L, I, V, M, S, or, T at position 2, and positivelycharged residues, such as R or K at the C-terminal position (in position9 of 9-mers). Exemplary members of the corresponding HLA family of HLAmolecules (the HLA-A3 superfamily) that bind the A3 supermotif include:A3 (A*0301), A11 (A*1101), A31 (A*3101), A*3301, and A*6801. Otherallele-encoded HLA molecules predicted to be members of the A3superfamily include A34, A66, and A*7401. As explained in detail below,peptide binding to each of the individual allele-specific HLA proteinscan be modulated by substitutions of amino acids at the primary and/orsecondary anchor positions of the peptide.

Representative peptide epitopes that contain the A3 supermotif are setforth on the attached Table VIII.

IV.C.2.d) HLA-A24 Supermotif

The HLA-A24 supermotif is characterized by the presence in peptideligands of an aromatic (F, W, or Y) residue as a primary anchor inposition 2 and a hydrophobic (Y, F, L, I, V, or M) residue as primaryanchor at the C-terminal position. The corresponding family of HLAmolecules that bind to the A24 supermotif (the A24 supertype) includesA*2402, A*3001, and A*2301. Peptide binding to each of theallele-specific HLA molecules can be modulated by substitutions atprimary anchor positions.

Representative peptide epitopes that contain the A24 supermotif are setforth on the attached Table IX.

IV.C.2.e) HLA-B7 Supermotif

The HLA-B7 supermotif is characterized by peptides bearing proline inposition 2 as a primary anchor and hydrophobic or aliphatic amino acids(L, I, V, M, A, F, W, or Y) as the primary anchor at the C-terminalposition. The corresponding family of HLA molecules that bind the B7supermotif (the HLA-B7 supertype) is comprised of at least a dozen HLA-Bproteins including B7, B*3501-1, B*3502-2, B*3501-3, B51, B*5301,B*5401, B*5501, B*5401, B*5501, B*5502, B*5601, B*6701, and B*7801 (See,e.g., Sidney, et al., J. Immunol. 154:247 (1995); Barber, et al., Curr.Biol. 5:179 (1995); Hill, et al., Nature 360:434 (1992); Rammensee, etal., Immunogenetics 41:178 (1995)). As explained in detail below,peptide binding to each of the individual allele-specific HLA proteinscan be modulated by substitutions at the primary and/or secondary anchorpositions of the peptide.

Representative peptide epitopes that contain the B7 supermotif are setforth on the attached Table X.

IV.C.2.f) HLA-B27 Supermotif

The HLA-B27 supermnotif is characterized by the presence in peptideligands of positively charged (R, H, or K) residues as primary anchorsat position 2 and hydrophobic (A, L, I, V, M, Y, F, or W) residues asprimary anchors at the C-terminal. Exemplary members of thecorresponding HLA molecules that bind to the B27 supermotif (the B27supertype) include B*14, B*1509, B*38, B*3901, B*3902, B*73, and variousB27 subtypes. Peptide binding to each of the allele-specific HLAmolecules can be modulated by substitutions at primary anchor positions.

Representative peptide epitopes that contain the B27 supermotif are setforth on the attached Table XI.

IV.C.2.g) HLA-B44 Supermotif

The HLA-B44 supermotif is characterized by the presence in peptideligands of negatively charged (D or E) residues as a primary anchor inposition 2, and hydrophobic residues (F, W, Y, L, I, M V, or A) as aprimary anchor at the C-terminal. Exemplary members of the correspondingfamily of HLA molecules that bind to the B44 supermnotif (the B44supertype) include B*3701, B*4402, B*4403, B60, and B61. Peptide bindingto each of the allele-specific HLA molecules can be modulated bysubstitutions at primary anchor positions.

Representative peptide epitopes that contain the B44 supermotif are setforth on the attached Table XII.

IV.C.2.h) HLA-B58 Supermotif

The HLA-B58 supermotif is characterized by the presence in peptideligands of small aliphatic residues (A, S, or T) as primary anchorresidues at position 2 and aromatic or hydrophobic residues (F, W, Y, L,I, or V) as primary anchor residues at the C-terminal. Exemplary membersof the corresponding HLA molecules that bind to the B58 supermotif (theB58 supertype) include B*1516, B*1517, B*5701, B*5702, and B*58. Peptidebinding to each of the allele-specific HLA molecules can be modulated bysubstitutions at primary anchor positions.

Representative peptide epitopes that contain the B58 supermotif are setforth on the attached Table XIII.

IV.C.2.i) HLA-B62 Supermotif

The HLA-B62 supermotif is characterized by the presence in peptideligands of the polar aliphatic residue Q or the hydrophobic aliphaticresidues (L, V, M, or I) as a primary anchor in position 2 andhydrophobic residues (F, W, Y, M, I, or V) as a primary anchor at theC-terminal position. Exemplary members of the corresponding HLAmolecules that a bind to the B62 supermotif (the B62 supertype) includeB46, B52, B62, B75, and B77. Peptide binding to each of theallele-specific HLA molecules can be modulated by substitutions atprimary anchor positions.

Representative peptide epitopes that contain the B62 supermotif are setforth on the attached Table XIV.

IV.C.2.j) HLA-A1 Motif

The allele-specific HLA-A1 motif is characterized by the presence inpeptide ligands of T, S, or M as a primary anchor residue at position 2and the presence of Y as a primary anchor residue at the C-terminalposition. Alternatively, a primary anchor residue may be present atposition 3 rather than position 2. This motif is characterized by thepresence of D, E, A, or S as a primary anchor residue in position 3 anda Y as a primary anchor residue at the C-terminus. Peptide binding toHLA A1 can be modulated by substitutions at primary and/or secondaryanchor positions.

Representative peptide epitopes that contain the A1 motif are set forthon the attached Table XV.

IV.C.2.k) HLA-A3 Motif

The allele-specific HLA-A3 motif is characterized by the presence inpeptide ligands of L, M, V, I, S, A, T, F, C, G, or D as a primaryanchor residue at position 2 and the presence of K, Y, R, H, F, or A asthe primary anchor residue at the C-terminal position. Peptide bindingto HLA-A3 can be modulated by substitutions at primary and/or secondaryanchor positions.

Representative peptide epitopes that contain the A3 motif are set forthon the attached Table XVI.

IV.C.2.1) HLA-A11 Motif

The allele-specific HLA-A11 motif is characterized by the presence inpeptide ligands of V, T, M, L, I, S, A, G, N, C, D, or F as a primaryanchor residue in position 2 and K, R, Y, or H as a primary anchorresidue at the C-terminal position. Peptide binding to HLA-A 11 can bemodulated by substitutions at primary and/or secondary anchor positions.

Representative peptide epitopes that contain the A11 motif are set forthon the attached Table XVI; peptides bearing the A3 allele-specific motifare also present in Table XVII. The A11 and A3 motifs have a number ofanchor residues in common, separate tables would provide a number ofredundant entries.

IV.C.2.m) HLA-A24 Motif

The allele-specific HLA-A24 motif is characterized by the presence inpeptide ligands of Y, F, W, or M as a primary anchor residue in position2 and F, L, I, or W as a primary anchor residue at the C-terminalposition. Peptide binding to HLA-A24 molecules can be modulated bysubstitutions at primary and/or secondary anchor positions.

Representative peptide epitopes that contain the A24 motif are set forthon the attached Table XVII.

IV.C.2.n) HLA-A2.1 Motif

The allele-specific HLA-A2.1 motif was first determined to becharacterized by the presence in peptide ligands of L, M, V, I, A or Tas a primary anchor residue in position 2 and, L, V, I, A, or T as aprimary anchor residue at the C-terminal position. The preferred andtolerated residues that characterize the primary anchor positions of theHLA-A2.1 motif are identical to the preferred residue of the A2supermotif. Secondary anchor residues that characterize the A2.1 motifhave additionally been defined as disclosed herein. These are disclosedin Table II. Peptide binding to HLA-A2.1 molecules can be modulated bysubstitutions at primary and/or secondary anchor positions.

Representative peptide epitopes that contain the A2.1 motif are setforth on the attached Table VII. These peptides, which bear the HLA-A2supermotif, also contain secondary anchor residues that arecharacteristic of the HLA-A2.1 motif. In one embodiment, the peptideepitope does not bear an L or M at position 2 and V at the C-terminalposition 9 of a 9-amino acid peptide.

The primary anchor residues of the HLA class I peptide epitopesupermotifs and motifs delineated above are summarized in Table I.Primary and secondary anchor positions are summarized in Table II.

Motifs Indicative of Class II HTL Inducing Peptide Epitopes

IV.C.2.o) HLA DR-1-4-7 Supermotif

Motifs have also been identified for peptides that bind to three commonHLA class II types, HLA DRB1*0401, DRB1*0101, and DRB1*0701. Peptidesbinding to these DR molecules carry a motif characterized by a largearomatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, or M)and a small, non-charged residue in position 6 (S, T, C, AP, V, I, L, orM). Allele specific secondary effects and secondary anchors for each ofthese HLA types have also been identified. These are set forth in TableIII. Peptide binding to HLA-DR4, DR1, and DR7 can be modulated bysubstitutions at primary and/or secondary anchor positions.

Representative peptides are set forth in Table XVIII.

IV.C.2.p) HLA DR3 Motifs

Two alternative motifs characterize peptides that bind to HLA-DR3molecules. In the first motif, a large, hydrophobic residue (I, L, V, M,Y, or F) is present in anchor position 1 and D is present as an anchorat position 4, which is defined as being 3 positions from anchorposition 1 towards the carboxyl terminus regardless of the location ofanchor position 1 in the peptide. Lack of either the large, hydrophobicresidue at anchor position 1, or of the negatively charged or amide-likeanchor residue at position 4 may be compensated for by the presence of apositive charge at position 6 (which is defined as being 5 positionsfrom anchor position 1 towards the carboxyl terminus). Thus for thesecond, alternative motif I, L, V, M, Y, F, or A is present at anchorposition 1; D, N, Q, E, S, or T is present at anchor position 4; and K,R, or H is present at anchor position 6. Peptide binding to HLA-DR3 canbe modulated by substitutions at primary and/or secondary anchorpositions.

Representative peptides are set forth in Table IXX.

IV.C.3. Enhancing Population Coverage of the Vaccine

Vaccines that have broad population coverage are preferred because theyare more commercially viable and generally applicable to the mostpeople. Broad population coverage can be obtained using the peptides ofthe invention (and nucleic acid compositions that encode such peptides)through selecting peptide epitopes that bind to HLA alleles which, whenconsidered in total, are present in most of the population. Table XXlists the overall frequencies of the A2-, A3-, and B7-supertypes invarious ethnicities. Coverage in excess of 80% is achieved with thesemotifs. These results suggest that effective and non-ethnically biasedpopulation coverage is achieved upon use of a limited number ofcross-reactive peptides. Although the population coverage reached withthese three main peptide specificities is high, coverage can be expandedto reach 95% population coverage and above, and more easily achievetruly multispecific responses upon use of additional supermotif orallele-specific motif bearing peptides.

Table XX summarizes the HLA supertypes that have been identified, andindicates an estimate of their combined prevalence in major ethnicgroups. The B44-, A1-, and A24-supertypes are present, on average, inover 25% of the world's major ethnic populations. While less prevalentoverall, the B27-, B58-, and B62 supertypes are each present with afrequency >25% in at least one major ethnic group. The Table indicatesthe population coverage achieved by the A2-, A3-, and B7-supertypes, andthe incremental coverage obtained by the addition of A1-, A24-, andB44-supertypes, or all of the supertypes described herein. As shown, byincluding epitopes from the six most frequent supertypes, an averagepopulation coverage of 99% is obtained for five major ethnic groups.

The data presented herein, together with the previous definition of theA2-, A3-, and B7-supertypes, indicates that all antigens, with thepossible exception of A29, B8, and B46, can be classified into a totalof nine HLA supertypes. Focusing on the six most common supertypesaffords population coverage greater than 98% for all major ethnicpopulations.

IV.D. Immune Response Stimulating Peptide Analogs

Although peptides with suitable cross-reactivity among all alleles of asuperfamily are identified by the screening procedures described above,cross-reactivity is not always complete and in such cases procedures tofurther increase cross-reactivity of peptides can be useful; suchprocedures can also be used to modify other properties of the peptides.Having established the general rules that govern cross-reactivity ofpeptides for HLA alleles within a given motif or supermotif,modification (i.e., analoging) of the structure of peptides ofparticular interest in order to achieve broader (or otherwise modified)HLA binding capacity can be performed. More specifically, peptides whichexhibit the broadest cross-reactivity patterns, (both amongst the knownT cell epitopes, as well as the more extended set of peptides thatcontain the appropriate supermotifs), can be produced in accordance withthe teachings herein.

The strategy employed utilizes the motifs or supermotifs which correlatewith binding to certain HLA molecules. The motifs or supermotifs aredefined by having primary anchors, though secondary anchors can also bemodified. Analog peptides can be created by substituting amino acidsresidues at primary anchor, secondary anchor, or at primary andsecondary anchor positions. Generally, analogs are made for peptidesthat already bear a motif or supermotif. Preferred secondary anchorresidues of supermotifs and motifs that have been defined for HLA classI and class II binding peptides are shown in Tables II and III,respectively.

For a number of the motifs or supermotifs in accordance with theinvention, residues are defined which are deleterious to binding toallele-specific HLA molecules or members of HLA supertypes that bind tothe respective motif or supermotif (Tables II and III). Accordingly,removal of residues that are detrimental to binding can be performed inaccordance with the present invention. For example, in the case of theA3 supertype, when all peptides that have such deleterious residues areremoved from the population of analyzed peptides, the incidence ofcross-reactivity increases from 22% to 37% (see, e.g., Sidney, J. etal., Hu. Immunol. 45:79, 1996). Thus, one strategy to improve thecross-reactivity of peptides within a given supermotif is simply todelete one or more of the deleterious residues present within a peptideand substitute a small “neutral” residue such as Ala (that may notinfluence T cell recognition of the peptide). An enhanced likelihood ofcross-reactivity is expected if, together with elimination ofdetrimental residues within a peptide, residues associated with highaffinity binding to multiple alleles within a superfamily are inserted.

To ensure that changes in the native or original epitope recognized by Tcells do not lead to a failure of killing antigen presenting cellspresenting the unaltered “wild type” peptide (or, in the case of classII epitopes, a failure to elicit helper T cells that cross-react withthe wild type peptides), the variant peptide may be used to immunize Tcells in vitro from individuals of the appropriate HLA allele, and thecells' capacity to induce lysis of wild type peptide sensitized targetcells is evaluated. In both class I and class II systems it will bedesirable to use as targets, cells that have been either infected ortransfected with the appropriate genes to establish whether endogenouslyproduced antigen is also recognized by the relevant T cells.

Another embodiment of the invention to ensure adequate numbers ofcross-reactive cellular binders is to create analogs of weak bindingpeptides. Class I peptides exhibiting binding affinities of 500-50000nM, and carrying an acceptable but suboptimal primary anchor residue atone or both positions can be “fixed” by substituting preferred anchorresidues in accordance with the respective supertype. The analogpeptides can then be tested for crossbinding activity.

Another embodiment for generating effective peptide analogs involves thesubstitution of residues that have an adverse impact on peptidestability or solubility in a liquid environment. This substitution mayoccur at any position of the peptide epitope. For example, a cysteine(C) can be substituted out in favor of α-amino butyric acid. Due to itschemical nature, cysteine has the propensity to form disulfide bridgesand sufficiently alter the peptide structurally so as to reduce bindingcapacity. Substituting α-amino butyric acid for C not only alleviatesthis problem, but actually improves binding and crossbinding capabilityin certain instances (Review: A. Sette et al, In: Persistent ViralInfections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, inpress, 1998). Substitution of cysteine with α-amino butyric acid mayoccur at any residue of a peptide epitope, i.e. at either anchor ornon-anchor positions.

In general, CTL and HTL responses are not directed against all possibleepitopes. Rather, they are restricted to a few immunodominantdeterminants (Zinkemagel, et al., Adv. Immunol. 27:5159, 1979; Bennink,et al., J. Exp. Med. 168:19351939, 1988; Rawle, et al., J. Immunol.146:3977-3984, 1991). It has been recognized that immunodominance(Benacerraf, et al., Science 175:273-279, 1972) could be explained byeither the ability of a given epitope to selectively bind a particularHLA protein (determinant selection theory) (Vitiello, et al., J.Immunol. 131:1635, 1983); Rosenthal, et al., Nature 267:156-158, 1977),or being selectively recognized by the existing TCR (T cell receptor)specificity (repertoire theory) (Klein, J., IMMUNOLOGY, THE SCIENCE OFSELFNONSELF DISCRIMINATION, John Wiley & Sons, N.Y., pp. 270-310, 1982).It has been demonstrated that additional factors, mostly linked toprocessing events, can also play a key role in dictating, beyond strictimmunogenicity, which of the many potential determinants will bepresented as immunodominant (Sercarz, et al., Annu. Rev. Immunol.11:729-766, 1993).

The concept of dominance and subdominance is relevant to immunotherapyof both infectious diseases and cancer. For example, in the course ofchronic viral disease, recruitment of subdominant epitopes can beimportant for successful clearance of the infection, especially ifdominant CTL or HTL specificities have been inactivated by functionaltolerance, suppression, mutation of viruses and other mechanisms(Franco, et al., Curr. Opin. Immunol. 7:524-531, (1995)). In the case ofcancer and tumor antigens, CTLs recognizing at least some of the highestbinding affinity peptides might be functionally inactivated. Lowerbinding affinity peptides are preferentially recognized at these times.

In particular, it has been noted that a significant number of epitopesderived from known non-viral tumor associated antigens (TAA) bind HLAclass I with intermediate affinity (IC₅₀ in the 50-500 nM range). Forexample, it has been found that 8 of 15 known TAA peptides recognized bytumor infiltrating lymphocytes (TIL) or CTL bound in the 50-500 nMrange. (These data are in contrast with estimates that 90% of knownviral antigens that were recognized as peptides bound HLA with IC₅₀ of50 nM or less, while only approximately 10% bound in the 50-500 nM range(Sette, et al., J. Immunol., 153:558-5592 (1994)). In the cancer settingthis phenomenon is probably due to elimination, or functional inhibitionof the CTL recognizing several of the highest binding peptides,presumably because of T cell tolerization events.

Without intending to be bound by theory, it is believed that because Tcells to dominant epitopes may have been clonally deleted, selectingsubdominant epitopes may allow extant T cells to be recruited, whichwill then lead to a therapeutic response. However, the binding of HLAmolecules to subdominant epitopes is often less vigorous than todominant ones. Accordingly, there is a need to be able to modulate thebinding affinity of particular immunogenic epitopes for one or more BLAmolecules, and thereby to modulate the immune response elicited by thepeptide. Thus a need exists to prepare analog peptides which elicit amore vigorous response. This ability would greatly enhance theusefulness of peptide-based vaccines and therapeutic agents.

Representative analog peptides are set forth in Table XXI. The Tableindicates the length and sequence of the analog peptide as well as themotif or supermotif, if appropriate. The information in the “FixedNomenclature” column indicates the residues substituted at the indicatedposition numbers for the respective analog.

IV.E. Computer Screening of Protein Sequences from Disease-RelatedAntigens for Supermotif or Motif Containing Peptides

Computer programs that allow the rapid screening of protein sequencesfor the occurrence of the subject supermotifs or motifs are encompassedby the present invention; as are programs that permit the generation ofanalog peptides. These programs are implemented to analyze anyidentified amino acid sequence or operate on an unknown sequence andsimultaneously determine the sequence and identify motif-bearingepitopes thereof; analogs can be simultaneously determined as well.Generally, the identified sequences will be from a pathogenic organismor a tumor-associated peptide. For example, the target moleculesconsidered herein include all of the HBV proteins (e.g. surface, core,polymerase, and X).

In cases where the sequence of multiple variants of the same targetprotein are available, peptides are also selected on the basis of theirconservancy. A presently preferred criterion for conservancy definesthat the entire sequence of a peptide be totally conserved in 75% of thesequences evaluated for a specific protein; this definition ofconservancy has been employed herein.

It is important that the selection criteria utilized for prediction ofpeptide binding are as accurate as possible, to correlate mostefficiently with actual binding. Prediction of peptides that bind, forexample, to HLA-A*0201, on the basis of the presence of the appropriateprimary anchors, is positive at about a 30% rate (Ruppert, J. et al.Cell 74:929, 1993). However, by analyzing an extensive peptide-HLAbinding database, the present inventors have developed a number ofallele specific polynomial algorithms that dramatically increase thepredictive value over identification on the basis of the presence ofprimary anchor residues alone. These algorithms take into account notonly the presence or absence of the correct primary anchors, but alsoconsider the positive or deleterious presence of secondary anchorresidues (to account for the impact of different amino acids atdifferent positions). The algorithms are essentially based on thepremise that the overall affinity (or AG) of peptide-HLA interactionscan be approximated as a linear polynomial function of the type:ΔG=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)

-   -   where a_(ij) is a coefficient that represents the effect of the        presence of a given amino acid (j) at a given position (i) along        the sequence of a peptide of n amino acids. An important        assumption of this method is that the effects at each position        are essentially independent of each other. This assumption is        justified by studies that demonstrated that peptides are bound        to HLA molecules and recognized by T cells in essentially an        extended conformation. Derivation of specific algorithm        coefficients has been described in Gulukota et al. (Gulukota, K.        et al., J. Mol. Biol. 267:1258, 1997).

Additional methods to identify preferred peptide sequences, which alsomake use of specific motifs, include the use of neural networks andmolecular modeling programs (Gulukota, K. et al., J. Mol. Biol.267:1258, 1997; Milik et al., Nature Biotechnology 16:753, 1998; Altuviaet al., Hum. Immunol. 58:1, 1997; Altuvia et al, J. Mol. Biol. 249:244,1995).

For example, it has been shown that in sets of A*0201 motif peptides,69% of the peptides containing at least one preferred secondary anchorresidue while avoiding the presence of any deleterious secondary anchorresidues, will bind A*0201 with an IC₅₀ less than 500 nM (Ruppert, J. etal. Cell 74:929, 1993). These algorithms are also flexible in thatcut-off scores may be adjusted to select sets of peptides with greateror lower predicted binding properties, as desired.

In utilizing computer screening to identify peptide epitopes, allprotein sequence or translated sequence may be analyzed using softwaredeveloped to search for motifs, for example the “FINDPATTERNS” program(Devereux, et al. Nucl. Acids Res. 12:387-395, 1984) or MotifSearch 1.4software program (D. Brown, San Diego, Calif.) to identify potentialpeptide sequences containing appropriate HLA binding motifs. Asappreciated by one of ordinary skill in the art a large array ofsoftware and hardware options are available which can be employed toimplement the motifs of the invention relative to known or unknownpeptide sequences. The identified peptides will then be scored usingcustomized polynomial algorithms to predict their capacity to bindspecific HLA class I or class II alleles.

In accordance with the procedures described above, HBV peptides andanalogs thereof that are able to bind HLA supertype groups orallele-specific BLA molecules have been identified (Tables VI-XIX; TableXI).

IV.F. Assays to Detect T-Cell Responses

Once HLA binding peptides are identified, they can be tested for theability to elicit a T-cell response. The preparation and evaluation ofmotif-bearing peptides are described in PCT publications WO 94/20127 andWO 94/03205. Briefly, peptides comprising epitopes from a particularantigen are synthesized and tested for their ability to bind to theappropriate HLA proteins in assays using, for example, purified HLAclass I molecules and radioiodonated peptides and/or cells expressingempty class I molecules (which lack peptide in their receptor) by, forinstance, immunofluorescent staining and flow microfluorimetry,peptide-dependent class I assembly assays, and inhibition of CTLrecognition by peptide competition. Those peptides that bind to theclass I molecule are further evaluated for their ability to serve astargets for CTLs derived from infected or immunized individuals, as wellas for their capacity to induce primary in vitro or in vivo CTLresponses that can give rise to CTL populations capable of reacting withselected target cells associated with a disease. Corresponding assaysare used for evaluation of HLA class II binding peptides.

Conventional assays utilized to detect CTL responses includeproliferation assays, lymphokine secretion assays, direct cytotoxicityassays, and limiting dilution assays. For example, antigen-presentingcells that have been incubated with a peptide can be assayed for theability to induce CTL responses in responder cell populations.Antigen-presenting cells can be normal cells such as peripheral bloodmononuclear cells or dendritic cells. Alternatively, mutant mammaliancell lines that are deficient in their ability to load class I moleculeswith internally processed peptides and that have been transfected withthe appropriate human class I gene may be used to test for the capacityof the peptide to induce in vitro primary CTL responses.

Peripheral blood lymphocytes may be used as the responder cell source ofCTL precursors. The appropriate antigen-presenting cells are incubatedwith peptide and the peptide-loaded antigen-presenting cells are thenincubated with the responder cell population under optimized cultureconditions. Positive CTL activation can be determined by assaying theculture for the presence of CTLs that kill radio-labeled target cells,both specific peptide-pulsed targets as well as target cells expressingendogenously processed forms of the HBV antigen from which the peptidesequence was derived.

More recently, a method has also been devised which allows directquantification of antigen-specific T cells by staining withFluorescein-labelled HLA tetrameric complexes (Altman, J. D. et al.,Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, J. D. et al., Science274:94, 1996). Other relatively recent technical developments includestaining for intracellular lymphokines, and interferon release assays orELISPOT assays. Tetramer staining, intracellular lymphokine staining andELISPOT assays all appear to be at least 10-fold more sensitive thanmore conventional assays (Lalvani, A. et al., J. Exp. Med. 186:859,1997; Dunbar, P. R. et al., Curr. Biol. 8:413, 1998; Murali-Krishna, K.et al., Immunity 8:177, 1998).

HTL activation may also be assessed using such techniques as T cellproliferation and secretion of lymphokines, e.g. IL-2.

Alternatively, immunization of HLA transgenic mice can be used todetermine immunogenicity of peptide epitopes. Several transgenic mousemodels including mice with human A2.1, A11, and B7 alleles have beencharacterized and others (e.g., transgenic mice for HLA-A1 and A24) arebeing developed. HLA-DR1 and HLA-DR3 mouse models have also beendeveloped. Additional transgenic mouse models with other HLA alleles maybe generated as necessary. Mice may be immunized with peptidesemulsified in Incomplete Freund's Adjuvant and the resulting T cellstested for their capacity to recognize peptide-pulsed target cells andtarget cells transfected with appropriate genes. CTL responses may beanalyzed using cytotoxicity assays described above. Similarly, HTLresponses may be analyzed using such assays as T cell proliferation orsecretion of lymphokines.

IV.G. Preparation of Peptides

Peptides in accordance with the invention can be prepared synthetically,by recombinant DNA technology, or from natural sources such as nativetumors or pathogenic organisms. Peptide epitopes may be synthesizedindividually or as polyepitopic peptides. Although the peptide willpreferably be substantially free of other naturally occurring host cellproteins and fragments thereof, in some embodiments the peptides may besynthetically conjugated to native fragments or particles.

The peptides in accordance with the invention can be a variety oflengths, and either in their neutral (uncharged) forms or in forms whichare salts. Peptides may be synthesized The peptides in accordance withthe invention are either free of modifications such as glycosylation,side chain oxidation, or phosphorylation; or they contain thesemodifications, subject to the condition that modifications do notdestroy the biological activity of the peptides as described herein.

Desirably, the peptide will be as small as possible while stillmaintaining substantially all of the biological activity of the largepeptide. When possible, it may be desirable to optimize HLA class Ibinding peptides of the invention to a length of about 8 to about 13amino acid residues, preferably 9 to 10. HLA class II binding peptidesmay be optimized to a length of about 6 to about 25 amino acids inlength, preferably to between about 13 and about 20 residues.Preferably, the peptides are commensurate in size with endogenouslyprocessed pathogen-derived peptides or tumor cell peptides that arebound to the relevant HLA molecules. Moreover, the identification andpreparation of peptides of other lengths can be carried out using thetechniques described herein (e.g., the disclosures regarding primary andsecondary anchor positions). However, it is also preferred to identify alarger region of a native peptide that encompasses one and preferablytwo or more epitopes in accordance with the invention. This sequence isselected on the basis that it contains the greatest number of epitopesper amino acid length. It is to be appreciated that epitopes can bepresent in a frame-shifted manner, e.g. a 10 amino acid long peptidecould contain two 9 amino acid long epitopes and one 10 amino acid longepitope; each epitope can be exposed and bound by an HLA molecule uponadministration of a plurality of such peptides. This larger, preferablymulti-epitopic, peptide can then be generated synthetically,recombinantly, or via cleavage from the native source.

The peptides of the invention can be prepared in a wide variety of ways.For the preferred relatively short size, the peptides can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce ChemicalCo. (1984). Further, individual peptides may be joined using chemicalligation to produce larger peptides.

Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes an immunogenic peptide of interest isinserted into an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression. These procedures are generally known in the art, asdescribed generally in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Thus,recombinant polypeptides which comprise one or more peptide sequences ofthe invention can be used to present the appropriate T cell epitope.

As the nucleotide coding sequence for peptides of the preferred lengthscontemplated herein can be synthesized by chemical techniques, forexample, the phosphotriester method of Matteucci, et al., J. Am. Chem.Soc. 103:3185 (1981) modification can be made simply by substituting theappropriate and desired nucleic acid base(s) for those that encode thenative peptide sequence. The coding sequence can then be provided withappropriate linkers and ligated into expression vectors commonlyavailable in the art, and the vectors used to transform suitable hoststo produce the desired fusion protein. A number of such vectors andsuitable host systems are now available. For expression of the fusionproteins, the coding sequence will be provided with operably linkedstart and stop codons, promoter and terminator regions and usually areplication system to provide an expression vector for expression in thedesired cellular host. For example, promoter sequences compatible withbacterial hosts are provided in plasmids containing convenientrestriction sites for insertion of the desired coding sequence. Theresulting expression vectors are transformed into suitable bacterialhosts. Of course, yeast, insect or mammalian cell hosts may also beused, employing suitable vectors and control sequences.

IV.H. Peptide Epitope Reagents to Evaluate Immune Responses.

HLA class I and class II binding peptides as described herein can beused, in one embodiment of the invention, as reagents to evaluate animmune response. The immune response to be evaluated may be induced byusing as an immunogen any agent that would potentially result in theproduction of antigen-specific CTLs or HTLs to the peptide epitope(s) tobe employed as the reagent. The peptide reagent is not used as theimmunogen.

For example, a peptide of the invention may be used in a tetramerstaining assay to assess peripheral blood mononuclear cells for thepresence of antigen-specific CTLs following exposure to a pathogen orimmunogen. The HLA-tetrameric complex is used to directly visualizeantigen-specific CTLs (see, e.g., Ogg et al. Science 279:2103-2106,1998; and Altman et al. Science 174:94-96, 1996) and determine thefrequency of the antigen-specific CTL population in a sample ofperipheral blood mononuclear cells. A tetramer reagent using a peptideof the invention may be generated as follows: A peptide that binds to anallele-specific HLA molecules, or supertype molecules, is refolded inthe presence of the corresponding HLA heavy chain and β₂-microglobulinto generate a trimolecular complex. The complex is biotinylated at thecarboxyl terminal end of the heavy chain at a site that was previouslyengineered into the protein. Tetramer formation is then induced by theaddition of streptavidin. By means of fluorescently labeledstreptavidin, the tetramer can be used to stain antigen-specific cells.The cells may then be identified, for example, by flow cytometry. Suchan analysis may be used for diagnostic or prognostic purposes.

Peptides of the invention may also be used as reagents to evaluateimmune recall responses. (see, e.g., Bertoni et al. J. Clin. Invest.100:503-513, 1997 and Penna et al. J. Exp. Med. 174:1565-1570, 1991.)For example, patient PBC samples from individuals with acute hepatitis Bor who have recently recovered from acute hepatitis B may be analyzedfor the presence of HBV antigen-specific CTLs using HBV-specificpeptides. A blood sample containing mononuclear cells may be evaluatedby cultivating the PBCs and stimulating the cells with a peptide of theinvention. After an appropriate cultivation period, the expanded cellpopulation may be analyzed for cytotoxic activity.

The peptides may also be used as reagents to evaluate the efficacy of avaccine. PBMCs obtained from a patient vaccinated with an immunogen maybe analyzed using, for example, either of the methods described above. Apatient is HLA typed, and appropriate peptide reagents that recognizeallele-specific molecules present in that patient may be selected forthe analysis. The immunogenicity of the vaccine will be indicated by thepresence of HBV epitope-specific CTLs in the PBMC sample.

IV.I. Vaccine Compositions

Vaccines that contain as an active ingredient an immunogenicallyeffective amount of one or more peptides as described herein are afurther embodiment of the invention. Once appropriately immunogenicepitopes have been defined, they can be sorted and delivered by variousmeans, herein referred to as “vaccine” compositions. Such vaccinecompositions can include, for example, lipopeptides (Vitiello, A. etal., J. Clin. Invest. 95:341, 1995), peptides compositions encapsulatedin poly(DL-lactide-co-glycolide) (PLG) microspheres (see, e.g.,Eldridge, et al. Molec. Immunol. 28:287-294, 1991: Alonso et al. Vaccine12:299-306, 1994; Jones et al. Vaccine 13:675-681, 1995), peptidecompositions encapsulated in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al. Nature 344:873-875, 1990; Hu et al. Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), viral delivery vectors(Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986;Hu, S. L. et al., Nature 320:537, 1986; Kieny, M. -P. et al., AIDSBio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148,1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viralor synthetic origin (Kofler, N. et al., J. Immunol. Methods. 192:25,1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D.,Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel,F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K.et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted, also know as receptor mediated targeting,delivery technologies also may be used such as those of AvantImmunotherapeutics, Inc. (Needham, Mass.).

Furthermore, vaccines in accordance with the invention encompasscompositions of one or more of the claimed peptide(s) that can beintroduced into a host, including humans, linked to its own carrier, oras a homopolymer or heteropolymer of active peptide units., Such apolymer has the advantage of increased immunological reaction and, wheredifferent peptides are used to make up the polymer, the additionalability to induce antibodies and/or CTLs that react with differentantigenic determinants of the pathogenic organism or tumor-relatedpeptide targetted for an immune response.

Furthermore, useful carriers that can be used with vaccines of theinvention are well known in the art, and include, e.g., thyroglobulin,albumins such as human serum albumin, tetanus toxoid, polyamino acidssuch as poly L-lysine, poly L-glutamic acid, influenza, hepatitis Bvirus core protein, hepatitis B virus recombinant vaccine and the like.The vaccines can contain a physiologically tolerable (i.e., acceptable)diluent such as water, or saline, preferably phosphate buffered saline.The vaccines also typically include an adjuvant. Adjuvants such asincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum are examples of materials well known in the art. Additionally, asdisclosed herein, CTL responses can be primed by conjugating peptides ofthe invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine P₃CSS).

As disclosed in greater detail herein, upon immunization with a peptidecomposition in accordance with the invention, via injection, aerosol,oral, transdermal, transmucosal, intrapleural, intrathecal, or othersuitable routes, the immune system of the host responds to the vaccineby producing large amounts of CTLs specific for the desired antigen, andthe host becomes at least partially immune to later infection, or atleast partially resistant to developing an ongoing chronic infection.

In some instances it may be desirable to combine the class I peptidevaccines of the invention with vaccines which induce or facilitateneutralizing antibody responses to the target antigen of interest,particularly to viral envelope antigens. A preferred embodiment of sucha composition comprises class I and class II epitopes in accordance withthe invention. An alternative embodiment of such a composition comprisesa class I and/or class II epitope in accordance with the invention,along with a PADRE®(Epimmune, San Diego, Calif.) molecule (described inthe related U.S. Ser. No. 08/485,218, which is a CIP of U.S. Ser. No.08/305,871, now U.S. Pat. No. 5,736,142, which is a CIP of abandonedapplication U.S. Ser. No. 08/121,101.) Furthermore, any of theseembodiments can be administered as a nucleic acid mediated modality.

For therapeutic or immunization purposes, the peptides of the inventioncan also be expressed by viral or bacterial vectors. Examples ofexpression vectors include attenuated viral hosts, such as vaccinia orfowlpox. This approach involves the use of vaccinia virus as a vector toexpress nucleotide sequences that encode the peptides of the invention.Upon introduction into an acutely or chronically infected host or into anon-infected host, the recombinant vaccinia virus expresses theimmunogenic peptide, and thereby elicits a host CTL and/or HTL response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover, et al.Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Antigenic peptides are used to elicit a CTL and/or HTL response ex vivo,as well. The resulting CTL or HTL cells, can be used to treat chronicinfections, or tumors in patients that do not respond to otherconventional forms of therapy, or will not respond to a therapeuticvaccine peptide or nucleic acid in accordance with the invention. Exvivo CTL or HTL responses to a particular pathogen (infectious agent ortumor antigen) are induced by incubating in tissue culture the patient'sCTL or HTL precursor cells together with a source of antigen-presentingcells (APC), such as dendritic cells, and the appropriate immunogenicpeptide. After an appropriate incubation time (typically about 14weeks), in which the precursor cells are activated, mature and expandinto effector cells, the cells are infused back into the patient, wherethey will destroy (CTL) or facilitate destruction (HTL) of theirspecific target cell (an infected cell or a tumor cell).

Transfected dendritic cells may also be used as antigen presentingcells. Alternatively, dendritic cells are transfected, e.g., with aminigene construct in accordance with the invention, in order to elicitimmune responses. Minigenes will be discussed in greater detail in afollowing section.

DNA or RNA encoding one or more of the peptides of the invention canalso be administered to a patient. This approach is described, forinstance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat.Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647;WO 98/04720; and in more detail below. Examples of DNA-based deliverytechnologies include “naked DNA”, facilitated (bupivicaine, polymers,peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) delivery.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition, or forselecting epitopes to be included in a vaccine composition and/or to beencoded by a minigene. It is preferred that each of the followingprinciples are balanced in order to make the selection.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with HBV clearance.For HLA Class I this includes 3-4 epitopes that come from at least oneantigen of HBV. In other words, it has been observed that in patientswho spontaneously clear HBV, that they had generated an immune responseto at least 3 epitopes on at least one HBV antigen. For HLA Class II asimilar rationale is employed; again 3-4 epitopes are selected from atleast one HBV antigen (see e.g., Rosenberg et al. Science278:1447-1450).

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, or for Class II an IC₅₀ of 1000 nM or less.

3.) Sufficient supermotif bearing peptides, or a sufficient array ofallele-specific motif bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenpreferred to select analogs. When selecting epitopes for infectiousdisease-related antigens it is often preferable to select nativeepitopes. Therefore, of particular relevance for infectious diseasevaccines (but for cancer-related vaccines as well), are epitopesreferred to as “nested epitopes.” Nested epitopes occur where at leasttwo epitopes overlap in a given peptide sequence. A peptide comprising“transcendent nested epitopes” is a peptide that has both HLA class Iand HLA class II epitopes in it.

When providing nested epitopes, it is preferable to provide a sequencethat has the greatest number of epitopes per provided sequence. Alimitation on this principle is to avoid providing a peptide that is anylonger than the amino terminus of the amino terminal epitope and thecarboxyl terminus of the carboxyl terminal epitope in the peptide. Whenproviding a longer peptide sequence, such as a sequence comprisingnested epitopes, it is important to screen the sequence in order toinsure that it does not have pathological or other deleteriousbiological properties.

5.) When creating a minigene, as disclosed in greater detail in thefollowing section, an objective is to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same as those employed when selecting apeptide comprising nested epitopes. Thus, upon determination of thenucleic acid sequence to be provided as a minigene, the peptide encodedthereby is analyzed to determine whether any “junctional epitopes” havebeen created. A junctional epitope is an actual binding epitope, aspredicted, e.g., by motif analysis. Junctional epitopes are to beavoided because the recipient may generate an immune response to thatepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

IV.I.1. Minigene Vaccines

A growing body of experimental evidence demonstrates that a number ofdifferent approaches are available which allow simultaneous delivery ofmultiple epitopes. Nucleic acids encoding the peptides of the inventionare a particularly useful embodiment of the invention. Epitopes forinclusion in a minigene are preferably selected according to theguidelines above. A preferred means of administering nucleic acidsencoding the peptides of the invention uses minigene constructs encodingone or multiple epitopes of the invention. The use of multi-epitopeminigenes is described below and in, e.g. An, L. and Whitton, J. L., .1J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822,1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al.,Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encodingnine dominant HLA-A*0201- and A11-restricted epitopes derived from thepolymerase, envelope, and core proteins of HBV and HIV, the PADRE®universal helper T cell (HTL) epitope, and an ER-translocating signalsequence was engineered. Immunization of HLA transgenic mice with thisplasmid construct resulted in strong CTL induction responses against thenine epitopes tested, similar to those observed with a lipopeptide ofknown immunogenicity in humans, and significantly greater thanimmunization in oil-based adjuvants. Moreover, the immunogenicity ofDNA-encoded epitopes in vivo correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat could be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, a ubiquitinationsignal sequence, a leader sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF) or costimulatory molecules.Helper (HTL) epitopes can be joined to intracellular targeting signalsand expressed separately from expressed CTL epitopes; this allowsdirection of the HTL epitopes to a cell compartment different than thatof the CTL epitopes. If required, this could facilitate more efficiententry of HTL epitopes into the HLA class II pathway, thereby improvingCTL induction. In contrast to HTL or CTL induction, specificallydecreasing the immune response by co-expression of immunosuppressivemolecules (e.g. TGF-β) may be beneficial in certain diseases).

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids can also be used in theformulation (see, e.g., as described by WO 93/24640; Mannino &Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833;WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413(1987). In addition, glycolipids, fusogenic liposomes, peptides andcompounds referred to collectively as protective, interactive,non-condensing (PINC) could also be complexed to purified plasmid DNA toinfluence variables such as stability, intramuscular dispersion, ortrafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes, respectively. For example, the plasmid DNA is introduced intoa mammalian cell line that is suitable as a target for standard CTLchromium release assays. The transfection method used will be dependenton the final formulation. Electroporation can be used for “naked” DNA,whereas cationic lipids allow direct in vitro transfection. A plasmidexpressing green fluorescent protein (GFP) can be co-transfected toallow enrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates production of HLA presentation ofminigene-encoded CTL epitopes.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS, IPfor lipid-complexed DNA). Twenty-one days after immunization,splenocytes are harvested and restimulated for 1 week in the presence ofpeptides encoding each epitope being tested. For CTL effector cells,assays are conducted for cytolysis of peptide-loaded, chromium-51labeled target cells using standard techniques. Lysis of target cellssensitized by HLA loading of peptides corresponding to minigene-encodedepitopes demonstrates DNA vaccine function for in vivo induction ofCTLs.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

IV.I.2. Combinations with Helper Pepides

The peptides of the present invention, or analogs thereof, which haveimmunostimulatory activity may be modified to provide desiredattributes, such as improved serum half life, or to enhanceimmunogenicity.

For instance, the ability of the peptides to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Particularly preferred immunogenic peptides/T helper conjugates arelinked by a spacer molecule. The spacer is typically comprised ofrelatively small, neutral molecules, such as amino acids or amino acidmimetics, which are substantially uncharged under physiologicalconditions. The spacers are typically selected from, e.g., Ala, Gly, orother neutral spacers of nonpolar amino acids or neutral polar aminoacids. It will be understood that the optionally present spacer need notbe comprised of the same residues and thus may be a hetero- orhomo-oligomer. When present, the spacer will usually be at least one ortwo residues, more usually three to six residues. Alternatively, the CTLpeptide may be linked to the T helper peptide without a spacer.

The immunogenic peptide may be linked to the T helper peptide eitherdirectly or via a spacer either at the amino or carboxy terminus of theCTL peptide. The amino terminus of either the immunogenic peptide or theT helper peptide may be acylated. The T helper peptides used in theinvention can be modified in the same manner as CTL peptides. Forinstance, they may be modified to include D-amino acids or be conjugatedto other molecules such as lipids, proteins, sugars and the like.Exemplary T helper peptides include tetanus toxoid 830-843, influenza307-319, and malarial circumsporozoite 382-398 and 378-389.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in the majority of the population. This can beaccomplished by selecting amino acid sequences that bind to many, most,or all of the HLA class II molecules. These are known as “looselyHLA-restricted” or “promiscuous” T helper sequences. Examples of aminoacid sequences that are promiscuous include sequences from antigens suchas tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:2572),Plasmodium falciparum CS protein at positions 378-398(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 2573), and Streptococcus 18kD proteinat positions 116 (GAVDSILGGVATYGAA; SEQ ID NO:2574). Other examplesinclude peptides bearing a DR 1-4-7 supermotif.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE® Epimmune, Inc., San Diego, Calif.)are designed on the basis of their binding activity to most HLA-DR(human HLA class II) molecules. For instance, a pan-DR-binding epitopepeptide having the formula: aKXVWANTLKAAa, where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanineor L-alanine (SEQ ID NO:2575), has been found to bind to most HLA-DRalleles, and to stimulate the response of T helper lymphocytes from mostindividuals, regardless of their HLA type.

T helper epitopes can also be modified to alter their biologicalproperties. For example, peptides presenting T helper epitopes cancontain D-amino acids to increase their resistance to proteases and thusextend their serum half-life. Also, the epitope peptides of theinvention can be conjugated to other molecules such as lipids, proteinsor sugars, or any other synthetic compounds, to increase theirbiological activity. Specifically, the T helper peptide can beconjugated to one or more palmitic acid chains at either the amino orcarboxyl termini.

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primescytotoxic T lymphocytes. Lipids have been identified as agents capableof priming CTL in vivo against viral antigens. For example, palmiticacid residues can be attached to the ε-and α-amino groups of a lysineresidue and then linked, e.g., via one or more linking residues such asGly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. Thelipidated peptide can then be administered either directly in a micelleor particle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic comprises palmitic acid attached toε- and α-amino groups of Lys, which is attached via linkage, e.g.,Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide. See, Deres, et al., Nature 342:561 (1989).Peptides of the invention can be coupled to P₃CSS, for example, and thelipopeptide administered to an individual to specifically prime a CTLresponse to the target antigen. Moreover, because the induction ofneutralizing antibodies can also be primed with P₃CSS-conjugatedepitopes, two such compositions can be combined to more effectivelyelicit both humoral and cell-mediated responses to infection.

In addition, additional amino acids can be added to the termini of apeptide to provide for ease of linking peptides one to another, forcoupling to a carrier support, or larger peptide, for modifying thephysical or chemical properties of the peptide or oligopeptide, or thelike. Amino acids such as tyrosine, cysteine, lysine, glutamic oraspartic acid, or the like, can be introduced at the C- or N-terminus ofthe peptide or oligopeptide, particularly class I peptides. However, itis to be noted that modification at the carboxyl terminus may, in somecases, alter binding characteristics of the peptide. In addition, thepeptide or oligopeptide sequences can differ from the natural sequenceby being modified by terminal-NH₂ acylation, e.g., by alkanoyl (C₁-C₂₀)or thioglycolyl acetylation, terminal-carboxylamidation, e.g., ammonia,methylamine, etc. In some instances these modifications may providesites for linking to a support or other molecule.

IV.J. Administration of Vaccines for Therapeutic or ProphylacticPurposes

The peptides of the present invention and pharmaceutical and vaccinecompositions of the invention are useful for administration to mammals,particularly humans, to treat and/or prevent HBV infection. Vaccinecompositions containing the peptides of the invention are administeredto a patient susceptible to or otherwise at risk for HBV infection toelicit an immune response against HBV antigens and thus enhance thepatient's own immune response capabilities. In therapeutic applications,compositions are administered to a patient in an amount sufficient toelicit an effective CTL response to the virus or tumor antigen and tocure or at least partially arrest or slow symptoms and/or complications.An amount adequate to accomplish this is defined as “therapeuticallyeffective dose.” Amounts effective for this use will depend on, e.g.,the particular composition administered, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician. Generally the dosage range for an initialimmunization (i.e., therapeutic or prophylactic administration) isbetween about 1.0 μg to about 5000 μg of peptide, typically betweenabout 10 μg to about 1000 μg, for a 70 kg patient, followed by boostingdosages of between about 1.0 μg to about 5000 μg of peptide pursuant toa boosting regimen over weeks to months depending upon the patient'sresponse and condition as determined by measuring specific CTL activityin the patient's blood. The peptides and compositions of the presentinvention may be employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

As noted above, the “CTL” peptides of the invention induce immuneresponses when contacted with a CTL specific to an epitope comprised bythe peptide. The manner in which the peptide is contacted with the CTLis not critical to the invention. For instance, the peptide can becontacted with the CTL either in vivo or in vitro. If the contactingoccurs in vivo, the peptide itself can be administered to the patient,or other vehicles, e.g., DNA vectors encoding one or more peptides,vital vectors encoding the peptide(s), liposomes and the like, can beused, as described herein.

For pharmaceutical compositions, the immunogenic peptides, or DNAencoding them, are generally administered to an individual alreadyinfected with HBV. The peptides or DNA encoding them can be administeredindividually or as fusions of one or more peptide sequences. Those inthe incubation phase or the acute phase of infection can be treated withthe immunogenic peptides separately or in conjunction with othertreatments, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of HBV infection. This is followed by boosting doses until atleast symptoms are substantially abated and for a period thereafter. Inchronic infection, loading doses followed by boosting doses may berequired.

Treatment of an infected individual with the compositions of theinvention may hasten resolution of the infection in acutely infectedindividuals. For those individuals susceptible (or predisposed) todeveloping chronic infection, the compositions are particularly usefulin methods for preventing the evolution from acute to chronic infection.Where susceptible individuals are identified prior to or duringinfection, the composition can be targeted to them, minimizing need foradministration to a larger population.

The peptide or other compositions as used for the treatment of chronicHBV infection and to stimulate the immune system to eliminatepathogen-infected cells in, e.g., persons who have not manifestedsymptoms of disease but who act as a disease vector. In this context, itis generally important to provide an amount of immuno-potentiatingpeptide in a formulation and mode of administration sufficient toeffectively stimulate a cytotoxic T cell response; compositions whichstimulate helper T cell responses can also be given in accordance withthis embodiment of the invention. Thus, for treatment of chronicinfection, a representative dose is in the range of about 1.0 μg toabout 5000 μg, preferably about 10 μg to 1000 μg, per 70 kg patientweight per dose. Immunizing doses followed by boosting doses atestablished intervals, e.g., from four weeks to six months, may berequired, possibly for a prolonged period of time to effectivelyimmunize an individual. In the case of chronic infection, administrationshould continue until at least clinical symptoms or laboratory testsindicate that the viral infection has been eliminated or substantiallyabated and for a period thereafter. The dosages, routes ofadministration, and dose schedules are adjusted in accordance withmethodologies known in the art.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, intrathecal, or local administration.Preferably, the pharmaceutical compositions are administered parentally,e.g., intravenously, subcutaneously, intradermally, or intramuscularly.Thus, the invention provides compositions for parenteral administrationwhich comprise a solution of the immunogenic peptides dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH-adjusting and buffering agents, tonicityadjusting agents, wetting agents, preservatives, and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, ie., from less than about 0.1%, usually ator at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

The peptides of the invention may also be administered via liposomes,which serve to target the peptides to a particular tissue, such aslymphoid tissue, or targeted selectively to infected cells, as well asincrease the half-life of the peptide composition. Liposomes includeemulsions, foams, micelles, insoluble monolayers, liquid crystals,phospholipid dispersions, lamellar layers and the like. In thesepreparations the peptide to be delivered is incorporated as part of aliposome, alone or in conjunction with a molecule which binds to, e.g.,a receptor prevalent among lymphoid cells, such as monoclonal antibodieswhich bind to the CD45 antigen, or with other therapeutic or immunogeniccompositions. Thus, liposomes either filled or decorated with a desiredpeptide of the invention can be directed to the site of lymphoid cells,where the liposomes then deliver the peptide compositions. Liposomes foruse in the invention are formed from standard vesicle-forming lipids,which generally include neutral and negatively charged phospholipids anda sterol, such as cholesterol. The selection of lipids is generallyguided by consideration of, e.g., liposome size, acid lability andstability of the liposomes in the blood stream. A variety of methods areavailable for preparing liposomes, as described in, e.g., Szoka, et al.,Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871,4,501,728,4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, preferably1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included, asdesired, as with, e.g., lecithin for intranasal delivery.

The vaccine compositions of the invention may also be used purely asprophylactic agents. Vaccine compositions containing the peptideepitopes of the invention are administered to a patient susceptible to,or otherwise at risk for, HBV infection to elicit an immune responseagainst HBV antigens and thus enhance the patient's own immune responsecapabilities following exposure to HBV. Generally the dosage range foran initial prophylactic immunization is between about 1.0 μg to about5000 μg of peptide, typically between about 10 μg to about 1000 μg, fora 70 kg patient. This is followed by boosting dosages of between about1.0 μg to about 5000 μg of peptide administered at defined intervalsfrom about four weeks to six months after the initial administration ofvaccine. The immunogenicity of the vaccine may be assessed by measuringspecific CTL activity in the patient's blood.

IV.K. Kits

The peptide and nucleic acid compositions of this invention can beprovided in kit form together with instructions for vaccineadministration. Typically the kit would include desired peptidecompositions in a container, preferably in unit dosage form andinstructions for administration. An alternative kit would include aminigene construct with desired nucleic acids of the invention in acontainer, preferably in unit dosage form together with instruction foradministration. Lymphokines such as IL-2 or IL-12 may also be includedin the kit. Other kit components that may also be desirable include, forexample, a sterile syringe, booster dosages, and other desiredexcipients.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters that can be changed or modified to yield alternativeembodiments in accordance with the invention.

V. EXAMPLES Example 1 HLA Class I Binding Assays

The following example of peptide binding to HLA-A3 supertype moleculesdemonstrates quantification of binding affinities of HLA class Ipeptides. Analogous binding assays can be performed for other peptidesthat bind class I or class II HLA molecules. Furthermore, binding assayscan be performed with peptides that are not motif-bearing.

For example, the affinity of peptides bearing an HLA-A3 supermotif wasdetermined as follows. Epstein-Barr virus (EBV)-transformed homozygouscell lines were used as sources of class I molecules. Cell linesinclude, e.g., GM3107 (A3, B7; Human Genetic Mutant Repository); BVR(A11, B35.3, Cw4; Human Genetic Mutant Repository); SPACH (A31, B62,Cw1/3; ASHI Repository Collection); LWAGS (A*3301, B14, and Cw8; ASHIRepository Collection) (Bodmer, et al., Hum. Immunol. 43:149, 1995), anda C1R transfectant characterized by Dr. Walter Storkus (University ofPittsburgh) for the isolation of A*6801. Cell lines were maintained aspreviously described (Sidney, et al., J. Immunol. 154:247 (1995); Sette,et al., Mol. Immunol. 31:813 (1994)).

Cell lysates were prepared and HLA class I molecules purified inaccordance with disclosed protocols (Sidney, et al., J. Immunol. 154:247(1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, cells werelysed at a concentration of 10⁸ cells/ml in 50 mM Tris-HCl, pH 8.5,containing 1% Nonidet P-40 (Fluka Biochemika, Buchs, Switzerland), 150mM NaCl, 5 mM EDTA, and 2 mM PMSF. The lysates were passed through 0.45μM filters and cleared of nuclei and debris by centrifugation at 10,000g for 20 minutes. HILA proteins were then purified by affinitychromatography. Columns of inactivated Sepharose CL 4B and Protein ASepharose were used as precolumns. The cell lysate was depleted of HLA-Band HLA-C proteins by repeated passage over Protein A Sepharose beadsconjugated with the anti-HLA(B,C) antibody B1.23.2 (Rebai, et al.,Tissue Antigens 22:107 (1983)). Typically two to four passages wererequired for effective depletion. Subsequently, the anti HLA(A,B,C)antibody W6/32 (Barnstable, et al., Cell 14:9 (1978)) was used tocapture HLA-A molecules. Protein purity, concentration, andeffectiveness of depletion steps were monitored by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Binding Assays

Quantitative assays for the binding of peptides to soluble class Imolecules on the basis of the inhibition of binding of a radiolabeledstandard probe peptide to detergent solubilized HLA molecules wereperformed as described in the literature (Kubo, et al., J. Immunol.152:3913 (1994); Kast, et al., J. Immunol. 152:3904 (1994); Sidney, etal., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813(1994); Ruppert, et al., Cell 74:929 (1993)). Briefly, 1-10 nM ofradiolabeled probe peptide, iodinated by the Chloramine-T method(Greenwood, et al., Biochem. J. 89:114 (1963)), was co-incubated at roomtemperature with various amounts of HLA in the presence of 1 μM humanβ₂-microglobulin (Scripps Laboratories, San Diego, Calif., USA) and acocktail of protease inhibitors. At the end of a two day incubationperiod, the percent of HLA-bound radioactivity was determined by sizeexclusion gel filtration chromatography on a TSK 2000 column.

The A3CON1 peptide (sequence KVFPYALINK; SEQ ID NO:2576) (Kubo, et al.,J. Immunol. 152:3913 (1994)) was used as the radiolabeled probe for theA3, A11, A31, and A*6801 assays. A T7Y analogue of HBVc ₁₄₁₋₁₅₁(sequence STLPETYVVRRL; SEQ ID No:2577) (Missale, et al., J. Exp. Med.177:751 (1993)) was used as the radiolabeled probe for the A*3301 assay.In the case of competitive assays, the concentration of peptide yielding50% inhibition of the binding of the radiolabeled probe peptide (IC₅₀)was calculated. Peptides were usually tested at one or two high doses,and the IC₅₀ of peptides yielding positive inhibition were determined insubsequent experiments, in which two to six further dilutions weretested, as necessary. To achieve a suitable signal, HLA concentrationsyielding approximately 15% binding of the radiolabled probe peptide wereused for all competitive inhibition assays. Under these conditions theconcentration of the labeled peptide is less than the concentration ofthe HLA molecule and the IC₅₀ is less than the concentration of the HLAmolecule, therefore the measured IC₅₀s are reasonable approximations ofthe true K_(D) values. Each competitor peptide was tested in two to fourcompletely independent experiments. As a positive control, in eachexperiment, the unlabeled version of the relevant radiolabeled probe wastested and its IC₅₀ measured. The average IC₅₀ of A3CON1 for the A3,A11, A31, and A*6801 assays were 11, 6, 18, and 8 nM, respectively. Theaverage IC₅₀ of the HBV_(c) 141-151 peptide in the A*3301 assay was 29nM.

Example 2 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Peptides by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in preparing highly cross-reactive native peptides,as demonstrated herein. Moreover, the definition of HLA motifs andsupermotifs also allows one to engineer highly cross-reactive epitopesby identifying residues within a native peptide sequence which can beanaloged, or “fixed”, to confer upon a peptide certain characteristics,e.g., greater cross-reactivity within the group of HLA molecules thatmake-up the supertype, and/or greater binding affinity for some or allof those HLA molecules Examples of analog peptides that exhibitmodulated binding affinity are provided.

Analogs representing primary anchor single amino acid residuessubstituted with I residues at the C-terminus of two different B7-likepeptides (HBV env 313 and HBV pol 541) were synthesized and tested fortheir B7-supertype binding capacity. It was found that the Isubstitution had an overall positive effect on binding affinity and/orcross-reactivity in both cases. In the case of HBV env 313 the 19 (I atC-terminal position 9) replacement was effective in increasingcross-reactivity from 4 to 5 alleles bound by virtue of an almost400-fold increase B*5401 binding affinity. In the case of HBV pol 541,increased cross-reactivity was similarly achieved by a substantialincrease in B*5401 binding. Also, significant gains in binding affinityfor B*0702, B51, and B*5301 were observed with the HBV pol 541 I9analog.

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides by identifying particular residues at secondaryanchor positions that are associated with such cross-reactiveproperties. Demonstrating this, the capacity of a second set of peptidesrepresenting discreet single amino acid substitutions at positions oneand three of five different B7-supertype binding peptides weresynthesized and tested for their B-7 supertype binding capacity. In 4/4cases the effect of replacing the native residue at position 1 with thearomatic residue F (an “F1” substitution) resulted in an increase incross-reactivity, compared to the parent peptide, and, in mostinstances, binding affinity was increased three-fold or better (TableXXII). More specifically, for HBV env 313, MAGE2 170, and HCV core 168complete supertype cross-reactivity was achieved with the F1substitution analogs. These gains were achieved by dramaticallyincreasing B*5401 binding affinity. Also, gains in affinity were notedfor other alleles in the cases of HCV core 168((B*3501 and B*5301) andMAGE2 170((B*3501, B51 and B*5301). Finally, in the case of MAGE3 196,the F1 replacement was effective in increasing cross-reactivity becauseof gains in B*0702 binding. An almost 70-fold increase in B51 bindingcapacity was also noted.

Two analogs were also made using the supermotif positive F substitutionat position three (an “F3” substitution). In both instances increases inbinding affinity and cross-reactivity were achieved. Specifically, inthe case of HBV pol 541, the F3 substitution was effective in increasingcross-reactivity by virtue of its effect on B*5401 binding. In the caseof MAGE3 196, complete supertype cross-reactivity was achieved byincreasing B*0702 and B*3501 binding capacity. Also, in the case ofMAGE3 196, it is notable that increases in binding capacity between 40-and 5000-fold were obtained for B*3501, B51, B*5301, and B*5401.

In conclusion, these data demonstrate that by the use of even singleamino acid substitutions, it is possible to increase the bindingaffinity and/or cross-reactivity of peptide ligands for HLA supertypemolecules.

Example 3 Induction Of HLA-Restricted CTL By Subcutaneous Priming WithHBV Peptide In Incomplete Freund's Adjuvant (IFA)

The immunogenicity of HLA class I binding peptides can be assessed invivo as described in, e.g., Sette et al. J. Immunol. 153:5586-5592(1994). This example illustrates such a procedure, whereby subcutaneousinjection of HBV peptide in Incomplete Freund's Adjuvant (IFA) can beused to induce HBV-specific CTL in mice that are transgenic for a humanHLA allele such as the human HLA-A11 allele.

Priming and In Vitro Restimulation: Mice that are transgenic forHLA-A11, (e.g. HLA-A11/Kb strain) are injected with 100 microliters ofan emulsion of purified HBV peptide in IFA. The purified peptidecomprises an A11 motif, and is selected from the preferred peptideslisted in Table XVI or, alternatively, may be an analog peptide. Thepeptide epitope (50 μg/mouse) and equimolar amounts of the helperepitope HBV core 128-140 (140 μg/mouse) are dissolved in PBS/5% DMSO,emulsified in IFA, and injected subcutaneously at the base of the tailof the transgenic mice. Eleven days after priming, splenocytes (5×10⁶cells/well in a 24-well plate) obtained from these animals arerestimulated with syngeneic irradiated LPS blasts (2×10⁶/well) coatedwith peptide.

LPS blasts from unprimed HLA-A11 transgenic mice are prepared 72 hoursbefore use by suspending splenocytes in medium containing LPS (25 μg/ml)and dextran sulfate (7 μg/ml). Coating is achieved by incubating 50 μgof peptide with 1.2×10⁶ LPS blasts in a volume of 0.4 ml of RPMI mediumsupplemented with 10% FCS for 1 hour at 37° C. The cells are washed onceand then co-cultured with splenocytes. After six days, effector cellsare assayed, as outlined for example in Example 5, for cytotoxicityagainst ⁵¹Cr-labeled 3A4-721.221-A11K^(b) target cells in the presenceof the peptide.

The effector cells (2×10⁶ cells/well) are re-stimulated at weeklyintervals. For the first re-stimulation, peptide-coated LPS blasts areused, followed by peptide-coated A11/K^(b) cells. Six days afaterre-stimulation, effector cells are assayed for cytotoxicity as above.

Example 4 Recognition of Generation of Endogenous Processed AntigensAfter Priming

This example determines that CTL induced by in vivo priming with peptide(as disclosed in Example 3) recognize endogenously synthesized antigens.

Effector cells from the procedure disclosed in Example 3 arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled 3A4-721.221-A11/K^(b) targetcells, in the absence or presence of peptide, and also tested on ⁵¹Crlabeled target cells bearing the endogenously synthesized antigen.

The result will demonstrate that CTL lines obtained from animals primedwith peptide epitope recognize endogenously synthesized HBV antigen.

Example 5 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs in transgenic mice by useof an HBV CTL/HTL peptide conjugate. An analagous study may be found inOseroff et al. Vaccine 16:823-833 (1998). The peptide composition cancomprise multiple CTL and/o HTL epitopes. Such a peptide composition cancomprise a lipidated HTL epitope conjugated to a preferred CTL epitopecontaining, for example, an A11 motif or an analog of that epitope.

Lipopeptides are prepared by coupling the appropriate fatty acid to theamino terminus of the resin bound peptide. A typical procedure is asfollows: A dichloromethane solution of a four-fold excess of apre-formed symmetrical anhydride of the appropriate fatty acid is addedto the resin and the mixture is allowed to react for two hours. Theresin is washed with dichloromethane and dried. The resin is thentreated with trifluoroacetic acid in the presence of appropriatescavengers [e.g. 5% (v/v) water] for 60 minutes at 20° C. Afterevaporation of excess trifluoroacetic acid, the crude peptide is washedwith diethyl ether, dissolved in methanol and precipitated by theaddition of water. The peptide is collected by filtration and dried.

Preparation of peptides for immunization: Peptide compositions aretypically resuspended in DMSO at a concentration of 20 mg/ml. Beforeuse, peptides are prepared at the required concentration by dilution insaline or the appropriate medium.

Immunization procedures: A11/K^(b) mice, which are transgenic for thehuman HLA A11 allele, are primed subcutaneously (base of the tail) with0.1 ml of peptide conjugate formulated in saline, or DMSO/saline. Sevendays after priming, splenocytes obtained from these animals arerestimulated with syngeneic irradiated LPS-activated lymphoblasts coatedwith peptide.

Media:

a. RPMI-1640 supplemented with 10% fetal calf serum (FCS) 2 mMGlutamine, 50 μg/ml Gentamicin and 5×10⁻⁵ M 2-mercaptoethanol serves asculture medium

b. RPMI-1640 containing 25 mM HEPES buffer and supplemented with 2%(FCS) is used as cell washing medium.

Cell lines: The 3A4-721.221-A11/K^(b) cell line is used as target cells.This cell line is an EBV transformed cell line that was mutagenized andselected to be Class I negative which was transfected with anHLA-A11/K^(b) gene.

LPS-activated lymphoblasts: Splenocytes obtained from transgenic miceare resuspended at a concentration of 1-1.5×10⁶/ml in culture mediumsupplemented with 25 μg/ml LPS and 7 μg/ml dextran sulfate in 75 cmtissue culture flasks. After 72 hours at 37° C., the lymphoblasts arecollected for use by centrifugation.

Peptide coating of lymphoblasts: Peptide coating of the LPS activatedlymphoblasts is achieved by incubating 30×10⁶ irradiated (3000 rads)lymphoblasts with 100 μg of peptide in 1 ml of R10 medium for 1 hr at37° C. Cells are then washed once and resuspended in culture medium atthe desired concentration.

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, the effector cells areharvested and assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0-1.5×10⁶) are incubatedat 37° C. in the presence of 200 μl of sodium ⁵¹Cr chromate. After 60minutes, cells are washed three times and resuspended in R10 medium.Peptide is added where required at a concentration of 1 μg/ml. For theassay, 104 ⁵¹Cr-labeled target cells are added to differentconcentrations of effector cells (final volume of 200 μl) in U-bottom96-well plates. After a 6 hour incubation period at 37° C., a 0.1 mlaliquot of supernatant is removed from each well and radioactivity isdetermined in a Micromedic automatic gamma counter. The percent specificlysis is determined by the formula: percent specificrelease=100×(experimental release−spontaneous release)/(maximumrelease−spontaneous release). To facilitate comparison between separateCTL assays run under the same conditions, % ⁵¹Cr release data isexpressed as lytic units/10⁶ cells. One lytic unit is arbitrarilydefined as the number of effector cells required to achieve 30% lysis of10,000 target cells in a 6 hour SiCr release assay. To obtain specificlytic units/10⁶, the lytic units/10⁶ obtained in the absence of peptideis subtracted from the lytic units/10⁶ obtained in the presence ofpeptide. For example, if 30% ⁵¹Cr release is obtained at the E:T of 50:1(i.e., 5×10⁵ effector cells for 10,000 targets) in the absence ofpeptide and 5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in thepresence of peptide, the specific lytic units would be:(1×10⁶(5×10⁴)−(1×10⁶(5×10⁵)=18 LU/10⁶.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation. Analyses similar to this may be performed to evaluate theimmunogenicity of peptide conjugates containing multiple CTL epitopesand/or multiple HTL epitopes. In accordance with these procedures it isfound that CTL and HTL responses are induced.

Example 7 Induction Of Specific CTL Response In Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes is set up as an IND Phase I, dose escalation study (5, 50and 500 μg) and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 subjects are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Thus, the vaccine is found to be both safe and efficacious.

Example 8 Phase II Trials in Patients Infected with HBV

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients (male and female) havingchronic HBV infection. A main objective of the trials is to determine aneffective dose and regimen for inducing CTLs in chronically infected HBVpatients, to establish the safety of inducing a CTL response in thesepatients, and to see to what extent activation of CTLs improves theclinical picture of chronically infected CTL patients, as manifested bya transient flare in alanine aminotransferase (ALT), normalization ofALT, and reduction in HBV DNA. Such a study is designed, for example, asfollows:

The studies are performed in multiple centers in the U.S. and Canada.The trial design is an open-label, uncontrolled, dose escalationprotocol wherein the peptide composition is administered as a singledose followed six weeks later by a single booster shot of the same dose.The dosages are 50, 500 and 5,000 micrograms per injection.Drug-associated adverse effects are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and include bothmales and females. The patients represent diverse ethnic backgrounds.All of them are infected with HBV for over five years and are HIV, HCVand HDV negative, but have positive levels of HBe antigen and HBsantigen.

The magnitude and incidence of ALT flares and the levels of HBV DNA inthe blood are monitored to assess the effects of administering thepeptide compositions. The levels of HBV DNA in the blood are an indirectindication of the progress of treatment. The vaccine composition isfound to be both safe and efficacious in the treatment of chronic HBVinfection.

Example 9 Selection of CTL and HTL Epitopes for Inclusion in anHBV-specific Vaccine

This example illustrates the procedure for the selection of peptideepitopes for vaccine compositions of the invention.

The following principles are utilized when selecting an array ofepitopes for inclusion in a polyepitopic composition, or for selectingepitopes to be included in a vaccine composition and/or to be encoded bya minigene. Each of the following principles are balanced in order tomake the selection.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with HBV clearance.For HLA Class I this includes 3-4 epitopes that come from at least oneantigen of HBV. In other words, it has been observed that in patientswho spontaneously clear HBV, that they had generated an immune responseto at least 3 epitopes on at least one HBV antigen. For HLA Class II asimilar rationale is employed; again 3-4 epitopes are selected from atleast one HBV antigen.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, or for Class II an IC₅₀ of 1000 nM or less.

3.) Sufficient supermotif bearing peptides, or a sufficient array ofallele-specific motif bearing peptides, are selected to give broadpopulation coverage. For example, epitopes are selected to provide atleast 80% population coverage. A Monte Carlo analysis, a statisticalevaluation known in the art, is employed to assess population coverage.

4.) When selecting epitopes for HBV antigens it is often preferable toselect native epitopes. Therefore, of particular relevance forinfectious disease vaccines, are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A peptide comprising “transcendent nestedepitopes” is a peptide that has both HLA class I and HLA class IIepitopes in it.

When providing nested epitopes, a sequence that has the greatest numberof epitopes per provided sequence is provided. A limitation on thisprinciple is to avoid providing a peptide that is any longer than theamino terminus of the amino terminal epitope and the carboxyl terminusof the carboxyl terminal epitope in the peptide. When providing a longerpeptide sequence, such as a sequence comprising nested epitopes, thesequence is screened in order to insure that it does not havepathological or other deleterious biological properties.

5.) When creating a minigene, as disclosed in greater detail in theExample 10, an objective is to generate the smallest peptide possiblethat encompasses the epitopes of interest. The principles employed aresimilar, if not the same as those employed when selecting a peptidecomprising nested epitopes. Thus, upon determination of the nucleic acidsequence to be provided as a minigene, the peptide encoded thereby isanalyzed to determine whether any “junctional epitopes” have beencreated. A junctional epitope is an actual binding epitope, aspredicted, e.g., by motif analysis. Junctional epitopes are to beavoided because the recipient may generate an immune response to thatepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

Peptide epitopes for inclusion in vaccine compositions are, for example,selected from those lsited in Table XXIII. A vaccine compositioncomprised of selected peptides, when administered, is safe, efficacious,and elicits an immune response similar in magnitude of an immuneresponse that clears an acute HBV infection.

Example 10 Construction of Minigene Multi-Epitope DNA Pslasmids

Expression plasmids have been constructed and evaluated as described,for example, in U.S. Ser. No. 60/085,751 filed May 15, 1998 and U.S.Ser. No. 09/078,904 filed May 13, 1998. The binding peptide epitopes andtheir positions for some of the plasmids described therein are shown inFIG. 1 as example of the orientation of peptide epitopes in minigeneconstructs. Such a plasmid may, for example, also include multiple CTLand HTL peptide epitopes. In the present example, HLA-A11 motif-bearingpeptides are used in conjunction with DR supermotif-bearing peptides.Preferred A11 epitopes are identified, for example, in Table XVI orTable XXI and peptide epitopes recognized by HLA DR molecules (TablesXVIII and XIX). Four class I A11 motif-bearing peptide epitopes oranalogs of those peptide epitopes derived from the same HBV antigen,e.g. the envelope protein, are selected as CTL epitopes. Four class IImotif-bearing peptide epitopes derived from the same antigen, e.g., theenvelope protein, are selected as HTL epitopes. These epitopes are thenincorporated into a minigene for expression in an expression vector.

This example illustrates the methods to be used for construction of sucha minigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

A pMin minigene DNA plasmid is constructed from an early generation DNAplasmid designated as pMin.0. This plasmid contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by a string of CTL and HTL epitopes selected in accordance withprinciples disclosed herein. The pMIN sequence encodes an open readingframe fused to the Myc and His antibody epitope tag coded for by thepcDNA 3.1 Myc-His vector.

Overlapping oligonucleotides, for example eight oligonucleotides,averaging approximately 70 nucleotides in length with 15 nucleotideoverlaps, are synthesized and HPLC-purified. The oligonucleotides encodethe selected peptide epitopes as well as appropriate linker nucleotides.The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For the first PCR reaction, 5 μg of each of two oligonucleotides areannealed and extended: Oligonucleotides 1+2, 3+4, 5+6, and 7+8 arecombined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mMKCL, 10 mM (NH₄)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1%Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfupolymerase. The full-length dimer products are gel-purified, and tworeactions containing the product of 1+2 and 3+4, and the product of 5+6and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct for 25 additional cycles. The full-length product isgel-purified and cloned into pCR-blunt (Invitrogen) and individualclones are screened by sequencing.

Example 11 The Plasmid Construct and the Degree to which it InducesImmunogenicity

The degree to which the plasmid construct prepared using the methodologyoutlined in Example 10 is able to induce immunogenicity is evaluatedthrough in vivo injections into mice and in vitro CTL culture andcytotoxicity assays as detailed e.g., in U.S. Ser. No. 60/085,751 filedMay 15, 1998. To assess the capacity of the pMin minigene construct toinduce CTLs in vivo, HLA-A11/K^(b) transgenic mice are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide.

Splenocytes from immunized animals are stimulated twice with each of thepeptide epitopes encoded in the minigene, then assayed forpeptide-specific cytotoxic activity in a ⁵¹Cr release assay. The resultsindicate the magnitude of the CTL response directed against each of itsA11-restricted epitopes, thus indicating the in vivo immunogenicity ofthe minigene vaccine. It is, therefore, found that the minigene elicitsimmune responses directed toward A11-restricted epitopes.

Example 12 Peptide Composition for Prophylactic Uses

Vaccine compositions of the present invention are used to prevent HBVinfection in persons who are at risk. For example, a polyepitopicpeptide epitope composition containing multiple CTL and HTL epitopessuch as those selected in Examples 9 and/or 10, which are also selectedto target greater than 80% of the population, is administered toindividuals at risk for HBV infection. The composition is provided as asingle lipidated polypeptide that encompasses multiple epitopes. Thevaccine is administered in an aqueous carrier comprised of FreundsIncomplete Adjuvant. The dose of peptide for the initial immunization isfrom about 1 to about 5,000 μg for a 70 kg patient. The initialadministration of vaccine is followed by booster dosages at 4 weeksfollowed by evaluation of the magnitude of the immune response in thepatient by techniques that determine the presence of epitope-specificCTL populations in a PBMC sample. Additional booster doses areadministered as required. The composition is found to be both safe andefficacious as a prophylaxis against HBV infection.

Alternatively, the polyepitopic peptide composition can be administeredas a nucleic acid in accordance with methodologies known in the art anddisclosed herein.

Example 13 Polyepitopic Vaccine Compositions Derived from Native HBVSequences

A native HBV polyprotein sequence is screened, preferably using computeralgorithms defined for each class I and/or class II supermotif or motif,to identify “relatively short” regions of the polyprotein that comprisemultiple epitopes. This relatively short sequence that contains multipledistinct, even overlapping, epitopes is selected and used to generate aminigene construct. The construct is engineered to express the peptide,which corresponds to the native protein sequence. The “relatively short”peptide is less than 100 amino acids in length, preferably less than 75amino acids in length, and more preferably less than 50 amino acids inlength. The protein sequence of the vaccine composition is selectedbecause it has maximal number of epitopes contained within the sequence.As noted herein, epitope motifs may be overlapping (i.e., frame shiftedrelative to one another) with frame shifted overlapping epitopes, e.g.two 9-mer epitopes can be present in a 10 amino acid peptide. Such avaccine composition is administered for therapeutic or prophylacticpurposes.

The vaccine composition will preferably include, for example, three CTLepitopes and at least one HTL epitope from the source antigen.Junctional sequences will be analyzed to avoid sequences containing apotentially immunodominant epitope. This polyepitopic native sequence isadministered either as a peptide or as a nucleic acid sequence whichencodes the peptide. Alternatively, an analog can be made of this nativesequence.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup that ispresently unknown. Furthermore, this embodiment directs the immuneresponse to sequences that are present in native HBV antigens. Lastly,the embodiment provides an economy of scale when producing nucleic acidvaccine compositions.

Related to this embodiment, computer programs can be derived whichidentify, in a target sequence, the greatest number of epitopes persequence length.

Example 14 Polyepitotpic Vaccine Compositions Directed to MultipleDiseases

The HBV peptide epitopes of the present invention are used inconjunction with peptide epitopes from target antigens related to one ormore other diseases, to create a vaccine composition that is useful forthe prevention or treatment of HBV as well as another disease. Examplesof other diseases include, but are not limited to, HIV, HCV, and HPV.

For example, a polyepitopic peptide composition comprising multiple CTLand HTL epitopes that target greater than 98% of the population may becreated for administration to individuals at risk for both HBV and HIVinfection. The composition can be provided as a single polypeptide thatincorporates the multiple epitopes from the various disease-associatedsources.

Example 15 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific CTL populations corresponding to HBV. Such ananalysis may be performed as described by Ogg et al., Science279:2103-2106, 1998. In the following example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) may be used for a cross-sectional analysis of,for example, HBV Env-specific CTL frequencies from untreated HLAA*0201-positive indiviuals at different stages of infection using an HBVEnv peptide containing an A2.1 extended motif. Tetrameric complexes aresynethesized as described (Musey et al., N. Engl. J. Med. 337:1267,1997). Briefly, purified HLA heavy chain (A2.1 in this example) andβ2-microglobulin are synthesized by means of a prokaryotic expressionsystem. The heavy chain is modified by deletion of thetransmembrane-cytosolic tail and COOH-terminal addition of a sequencecontaining a BirA enzymatic biotinylation site. The heavy chain,β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′triphosphate and magnesium. Streptavidin-phycoerythrinconjugate is added in a 1:4 molar ratio, and the tetrameric product isconcentrated to 1 mg/ml. The resulting product is referred to astetramer-phycoerythrin.

Approximately one million PBMCs are centrifuged at 300 g for 5 minutesand resuspended in 50 ul of cold phosphate-buffered saline. Tri-coloranalysis is performed with the tetramer-phycoerythrin, along withanti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramerand antibodies on ice for 30 to 60 min and then washed twice beforeformaldehyde fixaation. Gates are applied to contain >99.98% of controlsamples. Controls for the tetramers include both A*0201-negativeindividuals and A*0201-positive uninfected donors. The percentage ofcells stained with the tetramer is then determined by flow cytometry.The results indicate the number of cells in the PBMC sample that containepitope-restricted CTLs, thereby readily indicating the stage ofinfection with HBV or the status of exposure to HBV or to a vaccine thatelicits a protective response.

Example 16 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from infectionor who are chronically infected with HBV or who have been vaccinatedwith an HBV vaccine.

For example, the class I restricted CTL response of persons at risk forHBV infection who have been vaccinated may be analyzed. The vaccine maybe any HBV vaccine. PBMC are collected from vaccinated individuals andHLA typed. Appropriate peptide reagents that, are highly conserved and,optimally, bear supermotifs to provide cross-reactivity with multipleHLA supertype family members are then used for analysis of samplesderived from individuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. Synthetic peptide is added at 10 μg/ml to eachwell and recombinant HBc Ag is added at 1 μg/ml to each well as a sourceof T cell help during the first week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 ml of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimualted with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with uninfected control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with syntheticpeptide at 10 μM and labeled with 100 μCi of ⁵¹Cr (Amersham Corp.,Arlington Heights, Ill.) for 1 hour after which they are washed fourtimes with HBSS. Cytolytic activity is determined in a standard 4-h,split well ⁵¹Cr release assay using U-bottomed 96 well plates containing3,000 targets/well. Stimulated PBMC are tested at E/T ratios of 20-50:1on day 14. Percent cytotoxicity is determined from the formula: 100 x[(experimental release-spontaneous release)/maximum release-spontaneousrelease)]. Maximum release is determined by lysis of targets bydetergent (2% Triton X-100® Sigma Chemical Co., St. Louis, Mo.).Spontaneous release is <25% of maximum release for all experiments.

The results of such an analysis will indicate to what extentHLA-restricted CTL populations have been stimulated with the vaccine. Ofcourse, this protocol can also be used to monitor prior HBV exposure.

The above examples are provided to illustrate the invention but not tolimit its scope. For example, the human terminology for the MajorHistocompatibility Complex, namely HLA, is used throughout thisdocument. It is to be appreciated that these principles can be extendedto other species as well. Moreover, peptide epitopes have been disclosedin the related application U.S. Ser. No. 08/820,360, which waspreviously incorporated by reference. Thus, other variants of theinvention will be readily apparent to one of ordinary skill in the artand are encompassed by the appended claims. All publications, patents,and patent application cited herein are hereby incorporated by referencefor all purposes.

TABLE I POSITION POSITION POSITION 3 (Primary C Terminus 2 (PrimaryAnchor) Anchor) (Primary Anchor) SUPERMOTIF A1 T,I, L,V,M,S F,W,Y A2L,I,V,M, A,T,Q I,V, M,A,T,L A3 V,S,M,A, T,L,I R,K A24 Y,F, W,I,V,L,M,TF,I, Y,W,L,M B7 P V,I,L,F, M,W,Y,A B27 R,H,K F,Y,L, W,M,I B44 E, DF,W,Y,L,I,M,V,A B58 A,T,S F,W,Y, L,I,V B62 Q,L, I,V,M,P F,W,Y, M,I,VMOTIF A1 T,S,M Y A1 D,E, A,S Y A2.1 L,M, V,O,I,A,T V, L,I,M,A,T A3L,M,V,I,S,A,T,F, C,G,D K,Y,R, H,F,A A11 V,T,M,L,I,S,A,G,N, C,D,F K,R,Y,H A24 Y,F,W,M F,L,I,W A*3101 M,V,T, A,L,I,S R, K A*3301 M,V,A,L,F,I,S,T R,K A*6801 A,V,T, M,S,L,I R,K B*0702 P L,M,F, W,Y,A,I,V B*3501 PL,M,F,W,Y, I,V,A B51 P L,I,V,F, W,Y,A,M B*5301 P I,M,F,W,Y, A,L,V B*5401P A,T,I,V, L,M,F,W,Y Bold residues are preferred, italicized residuesare less preferred. A peptide is considered motif-bearing if it hasprimary anchors at each primary anchor position for a motif orsupermotif as specified in the above table.

TABLE II POSITION SUPER- MOTIFS 1 2 3 4 5 6 7 8 C-terminus A1$\frac{1{^\circ}\mspace{14mu}{Anchor}}{T,I,L,V,M,S}$$\frac{1{^\circ}\mspace{14mu}{Anchor}}{F,W,Y}$ A2$\frac{1{^\circ}\mspace{14mu}{Anchor}}{L,I,V,M,A,T,Q}$$\frac{1{^\circ}\mspace{14mu}{Anchor}}{L,I,V,M,A,T}$ A3 preferred$\frac{1{^\circ}\mspace{14mu}{Anchor}}{V,S,M,A,T,L,I}$ Y,F,W (4/5) Y,F,W(3/5) Y,F,W (4/5) P (4/5) $\frac{1{^\circ}\mspace{14mu}{Anchor}}{R,K}$deleterious D,E (3/5); D,E (4/5) P (5/5) A24$\frac{1{^\circ}\mspace{14mu}{Anchor}}{Y,F,W,I,V,L,M,T}$$\frac{1{^\circ}\mspace{14mu}{Anchor}}{F,I,Y,W,L,M}$ B7 preferred F,W,Y(5/5) L,I,V,M (3/5) $\frac{1{^\circ}\mspace{14mu}{Anchor}}{P}$ F,W,Y(4/5) F,W,Y (3/5)$\frac{1{^\circ}\mspace{14mu}{Anchor}}{V,I,L,F,M,W,Y,A}$ deleterious D,E(3/5); D,E (3/5) G (4/5) Q,N (4/5) D,E (4/5) P (5/5); G (4/5); A (3/5);Q,N (3/5) B27 $\frac{1{^\circ}\mspace{14mu}{Anchor}}{R,H,K}$$\frac{1{^\circ}\mspace{14mu}{Anchor}}{F,Y,L,W,M,I}$ B44$\frac{1{^\circ}\mspace{14mu}{Anchor}}{E,D}$$\frac{1{^\circ}\mspace{14mu}{Anchor}}{F,W,Y,L,I,M,V,A}$ B58$\frac{1{^\circ}\mspace{14mu}{Anchor}}{A,T,S}$$\frac{1{^\circ}\mspace{14mu}{Anchor}}{F,W,Y,L,I,V}$ B62$\frac{1{^\circ}\mspace{14mu}{Anchor}}{Q,L,I,V,M,P}$$\frac{1{^\circ}\mspace{14mu}{Anchor}}{F,W,Y,M,I,V}$ MOTIFS A1 9-merpreferred G,F,Y,W $\frac{1{^\circ}\mspace{14mu}{Anchor}}{S,T,M}$ D,E,AY,F,W P D,E,Q,N Y,F,W $\frac{1{^\circ}\mspace{14mu}{Anchor}}{Y}$deleterious D,E R,H,K,L,I,V,M A H A P A1 9-mer preferred G,R,H,KA,S,T,C,L,I,V, M $\frac{1{^\circ}\mspace{14mu}{Anchor}}{D,E,A,S}$G,S,T,C A,S,T,C L,I,V,M D,E $\frac{1{^\circ}\mspace{14mu}{Anchor}}{Y}$deleterious A R,H,K,D,E,P,Y D,E P,Q,N R,H,K P,G G,P F,W POSITION 9 orPOSITION: 1 2 3 4 5 6 7 8 C-terminus C-terminus A1 9-mer preferred Y,F,W$\frac{1{^\circ}\mspace{14mu}{Anchor}}{S,T,M}$ D,E,A,Q,N A Y,F,W,Q,NP,A,S,T,C G,D,E P $\frac{1{^\circ}\mspace{14mu}{Anchor}}{Y}$ deleteriousG,P R,H,K,G,L,I,V D,E R,H,K Q,N,A R,H,K, Y,F,W R,H,K A M A1 10-merpreferred Y,F,W S,T,C,L,I,V,M$\frac{1{^\circ}\mspace{14mu}{Anchor}}{D,E,A,S}$ A Y,F,W P,G G Y,F,W$\frac{1{^\circ}\mspace{14mu}{Anchor}}{Y}$ deleterious R,H,KR,H,K,D,E,P,Y P G P,R,H,K Q,N F,W A2.1 9-mer preferred Y,F,W$\frac{1{^\circ}\mspace{14mu}{Anchor}}{L,M,I,V,Q,A,T}$ Y,F,W S,T,C Y,F,WA P $\frac{1{^\circ}\mspace{14mu}{Anchor}}{V,L,I,M,A,T}$ deleteriousD,E,P D,E,R,K,H R,K,H D,E,R,K,H A2.1 10-mer preferred A,Y,F,W$\frac{1{^\circ}\mspace{14mu}{Anchor}}{L,M,I,V,Q,A,T}$ L,V,I,M G GF,Y,W,L, V,I,M $\frac{1{^\circ}\mspace{14mu}{Anchor}}{V,L,I,M,A,T}$deleterious D,E,P D,E R,K,H,A P R,K,H D,E,R, K,L,H R,K,H A3 preferredR,H,K $\frac{1{^\circ}\mspace{14mu}{Anchor}}{L,M,V,I,S,A,T,F,C,G,D}$Y,F,W P,R,H,K,K, Y,F,W A Y,F,W P$\frac{1{^\circ}\mspace{14mu}{Anchor}}{K,Y,R,H,F,A}$ deleterious D,E,PD,E A11 preferred A$\frac{1{^\circ}\mspace{14mu}{Anchor}}{V,T,L,M,I,S,A,G,N,C,D,F}$ Y,F,WY,F,W A Y,F,W Y,F,W P $\frac{1{^\circ}\mspace{14mu}{Anchor}}{K,R,Y,H}$deleterious D,E,P A G A24 9-mer preferred Y,F,W,R,H,K$\frac{1{^\circ}\mspace{14mu}{Anchor}}{Y,F,W,M}$ S,T,C Y,F,W Y,F,W$\frac{1{^\circ}\mspace{14mu}{Anchor}}{F,L,I,W}$ deleterious D,E,G D,E GQ,N,P D,E,R,H G A,Q,N K A24 10-mer preferred$\frac{1{^\circ}\mspace{14mu}{Anchor}}{Y,F,W,M}$ P YFWP P$\frac{1{^\circ}\mspace{14mu}{Anchor}}{F,L,I,W}$ deleterious G,D,E Q,NR,H,K D,E A Q,N D,E,A A3101 preferred R,H,K$\frac{1{^\circ}\mspace{14mu}{Anchor}}{M,V,T,A,L,I,S}$ Y,F,W P Y,F,WY,F,W A,P $\frac{1{^\circ}\mspace{14mu}{Anchor}}{R,K}$ deleterious D,E,PD,E A,D,E D,E D,E D,E A3301 preferred$\frac{1{^\circ}\mspace{14mu}{Anchor}}{M,V,A,L,F,I,S,T}$ Y,F,W A,Y,F,W$\frac{1{^\circ}\mspace{14mu}{Anchor}}{R,K}$ deleterious G,P D,E A6801preferred Y,F,W,S,T,C$\frac{1{^\circ}\mspace{14mu}{Anchor}}{A,V,T,M,S,L,I}$ Y,F,W,L,I,V, MY,F,W P $\frac{1{^\circ}\mspace{14mu}{Anchor}}{R,K}$ deleterious G,PD,E,G R,H,K A B0702 preferred R,H,K,F,W, Y$\frac{1{^\circ}\mspace{14mu}{Anchor}}{P}$ R,H,K R,H,K R,H,K R,H,K P,A$\frac{1{^\circ}\mspace{14mu}{Anchor}}{L,M,F,W,Y,A,I,V}$ deleteriousD,E,Q,N,P D,E,P D,E D,E G,D,E Q,N D,E B3501 preferred F,W,Y,L,I,V, M$\frac{1{^\circ}\mspace{14mu}{Anchor}}{P}$ F,W,Y$\frac{1{^\circ}\mspace{14mu}{Anchor}}{L,M,F,W,Y,I,V,A}$ deleteriousA,G,P G G B51 preferred L,I,V,M,F,W, Y$\frac{1{^\circ}\mspace{14mu}{Anchor}}{P}$ F,W,Y S,T,C F,W,Y G F,W,Y$\frac{1{^\circ}\mspace{14mu}{Anchor}}{L,I,V,F,W,Y,A,M}$ deleteriousA,G,P,D,E,R, H,K,S,T,C D,E G D,E,Q,N G,D,E B5301 preferred L,I,V,M,F,W,Y $\frac{1{^\circ}\mspace{14mu}{Anchor}}{P}$ F,W,Y S,T,C F,W,Y L,I,V,M,F,W,Y F,W,Y $\frac{1{^\circ}\mspace{14mu}{Anchor}}{I,M,F,W,Y,A,L,V}$deleterious A,G,P,Q,N G R,H,K,Q,N D,E B5401 preferred F,W,Y$\frac{1{^\circ}\mspace{14mu}{Anchor}}{P}$ F,W,Y,L, I,V,M L,I,V,MA,L,I,V,M F,W,Y,A,P$\frac{1{^\circ}\mspace{14mu}{Anchor}}{A,T,I,V,L,M,F,W,Y}$ deleteriousG,P,Q,N,D,E G,D,E,S, T,C R,H,K,D,E D,E Q,N,D,G,E D,E Italicized residuesindicate less preferred or “tolerated” residues. The information in thisTable II specific for 9-mers unless otherwise specified.

TABLE III POSITION MOTIFS

DR4 preferred F,M,Y,L,I,V,W M T I V,S,T,C,P, M,H M,H A,L,I,M deleteriousW R W,D,E DR1 preferred M,F,L,I,V,W,Y P,A,M,Q V,M,A,T, M A,V,Mdeleterious C C,H F,D C,W,D S,P,L,I,C G,D,E D DR7 preferredM,F,L,I,V,W,Y M W A I,V,M,S,A, M I,V C,T,P,L deleterious C G G,R,D N GDR Supermotif M,F,L,I,V,W,Y V,M,S,T,A, C,P,L,I DR3 MOTIFS

motif a L,I,V,M,F,Y D preferred motif b L,I,V,M,F,A,Y D,N,Q,E,S,T K,R,Hpreferred Italicized residues indicate less preferred or “tolerated”residues.

TABLE IV HLA Class I Standard Peptide Binding Affinity. STANDARD BINDINGSEQ STANDARD AFFINITY ID ALLELE PEPTIDE SEQUENCE (nM) NO: A*0101 944.02YLEPAIAKY 25 2486 A*0201 941.01 FLPSDYFPSV 5.0 2487 A*0202 941.01FLPSDYFPSV 4.3 2487 A*0203 941.01 FLPSDYFPSV 10 2487 A*0206 941.01FLPSDYFPSV 3.7 2487 A*0207 941.01 FLPSDYFPSV 23 2487 A*6802 1141.02FTQAGYPAL 40 2488 A*0301 941.12 KVFPYALINK 11 2489 A*1101 940.06AVDLYHFLK 6.0 2490 A*3101 941.12 KVFPYALINK 18 2489 A*3301 1083.02STLPETYVVRR 29 2491 A*6801 941.12 KVFPYALINK 8.0 2489 A*2401 979.02AYIDNYNKF 12 2492 B*0702 1075.23 APRTLVYLL 5.5 2493 B*3501 1021.05FPFKYAAAF 7.2 2494 B51 1021.05 FPFKYAAAF 5.5 2494 B*5301 1021.05FPFKYAAAF 9.3 2494 B*5401 1021.05 FPFKYAAAF 10 2494

TABLE V HLA Class II Standard Peptide Binding Affinity. Binding Nomen-Standard Affinity Allele clature Peptide Sequence (nM) SEQ ID NO:DRB1*0101 DR1 515.01 PKYVKQNTLKLAT 5.0 2495 DRB1*0301 DR3 829.02YKTIAFDEEARR 300 2496 DRB1*0401 DR4w4 515.01 PKYVKQNTLKLAT 45 2495DRB1*0404 DR4w14 717.01 YARFQSQTTLKQKT 50 2497 DRB1*0405 DR4w15 717.01YARFQSQTTLKQKT 38 2497 DRB1*0701 DR7 553.01 QYIKANSKFIGITE 25 2498DRB1*0802 DR8w2 553.01 QYIKANSKFIGITE 49 2498 DRB1*0803 DR8w3 553.01QYIKANSKFIGITE 1600 2498 DRB1*0901 DR9 553.01 QYIKANSKFIGITE 75 2498DRB1*1101 DR5w11 553.01 QYIKANSKFIGITE 20 2498 DRB1*1201 DR5w12 1200.05EALIHQLKINPYVLS 298 2499 DRB1*1302 DR6w19 650.22 QYIKANAKFIGITE 3.5 2500DRB1*1501 DR2w2β1 507.02 GRTQDENPVVHFFK 9.1 2501 NIVTPRTPPP DRB3*0101DR52a 511 NGQIGNDPNRDIL 470 2502 DRB4*0101 DRw53 717.01 YARFQSQTTLKQKT58 2503 DRB5*0101 DR2w2β2 553.01 QYIKANSKFIGITE 20 2504 The“Nomenclature” column lists the allelic designations used in TableXVIII.

TABLE VI HBV A01 SUPER MOTIF (With binding information) ConservancyFreq. Protein Position Sequence String Peptide Filed A*0101 SEQ ID NO:95 19 POL 521 AICSVVRRAF XIXXXXXXXF 1 95 19 NUC 54 ALRQAILCW XLXXXXXXW 280 16 ENV 108 AMQWNSTTF XMXXXXXXF 3 100 20 POL 166 ASFCGSPY XSXXXXXY26.0026 * 4 100 20 POL 166 ASFCGSPYSW XSXXXXXXXW 5 90 18 NUC 19 ASKLCLGWXSXXXXXW 6 85 17 NUC 19 ASKLCLGWLW XSXXXXXXXW 7 80 16 POL 822 ASPLHVAWXSXXXXXW 8 100 20 ENV 312 CIPIPSSW XIXXXXXW 9 100 20 ENV 312 CIPIPSSWAFXIXXXXXXXF 10 95 19 ENV 253 CLIFLLVLLDY XLXXXXXXXXY 26.0548 11 95 19 ENV239 CLRRFIIF XLXXXXXF 12 75 15 ENV 239 CLRRFIIFLF XLXXXXXXXF 13 95 19POL 523 CSVVRRAF XSXXXXXF 14 100 20 ENV 310 CTCIPIPSSW XTXXXXXXXW 15 9018 NUC 31 DIDPYKEF XIXXXXXF 16 85 17 NUC 29 DLLDTASALY XLXXXXXXXY1.0519 * 11.1000 17 95 19 ENV 196 DSWWTSLNF XSXXXXXXF 20.0120 18 95 19NUC 43 ELLSFLPSDF XLXXXXXXXF 19 95 19 NUC 43 ELLSFLPSDFF XLXXXXXXXXF 2095 19 POL 374 ESRLVVDF XSXXXXXF 21 95 19 POL 374 ESRLVVDFSQF XSXXXXXXXXF22 80 16 ENV 248 FILLLCLIF XIXXXXXXF 23 80 16 ENV 246 FLFILLLCLIFXLXXXXXXXXF 24 95 19 ENV 256 FLLVLLDY XLXXXXXY 26.0027 25 95 19 POL 658FSPTYKAF XSXXXXXF 26 90 18 X 63 FSSAGPCALRF XSXXXXXXXXF 27 100 20 ENV333 FSWLSLLVPF XSXXXXXXXF 20.0263 28 95 19 POL 656 FTFSPTYKAF XTXXXXXXXF20.0262 29 95 19 ENV 346 FVGLSPTVW XVXXXXXXW 30 95 19 POL 627 GLLGFAAPFXLXXXXXXF 20.0124 31 95 19 POL 509 GLSPFLLAQF XLXXXXXXXF 32 85 17 NUC 29GMDIDPYKEF XMXXXXXXXF 26.0372 33 95 19 NUC 123 GVWIRTPPAY XVXXXXXXXY1.0525 0.0017 34 75 15 POL 569 HLNPNKTKRW XLXXXXXXXW 35 80 16 POL 491HLYSHPIILGF XLXXXXXXXXF 36 85 17 POL 715 HTAELLAACF XTXXXXXXXF 37 95 19NUC 52 HTALRQAILCW XTXXXXXXXXW 38 100 20 POL 149 HTLWKAGILY XTXXXXXXXY1.0542 * 0.0300 39 100 20 ENV 249 ILLLCLIF XLXXXXXF 40 80 16 POL 760ILRGTSFVY XLXXXXXXY 1.0205 * 0.0017 41 90 18 ENV 188 ILTIPQSLDSWXLXXXXXXXXW 42 90 18 POL 625 IVGLLGFAAPF XVXXXXXXXXF 43 80 16 POL 503KIPMGVGLSPF XIXXXXXXXXF 44 85 17 NUC 21 KLCLGWLW XLXXXXXW 45 75 15 POL108 KLIMPARF XLXXXXXF 46 75 15 POL 108 KLIMPARFY XLXXXXXXY 1.0171 0.001747 80 16 POL 610 KLPVNRPIDW XLXXXXXXXW 48 85 17 POL 574 KTKRWGYSLNFXTXXXXXXXXF 49 95 19 POL 55 KVGNFTGLY XVXXXXXXY 1.0166 * 0.0680 50 95 19ENV 254 LIFLLVLLDY XIXXXXXXXY 1.0899 * 0.0084 51 100 20 POL 109 LIMPARFYXIXXXXXY 26.0028 52 85 17 NUC 30 LLDTASALY XLXXXXXXY 1.0155 * 25.0000 5380 16 POL 752 LLGCAANW XLXXXXXW 54 95 19 POL 628 LLGFAAPF XLXXXXXF 55100 20 ENV 378 LLPIFFCLW XLXXXXXXW 56 100 20 ENV 378 LLPIFFCLWVYXLXXXXXXXXY 26.0549 * 57 95 19 NUC 44 LLSFLPSDF XLXXXXXXF 58 95 19 NUC44 LLSFLPSDFF XLXXXXXXXF 59 90 18 POL 407 LLSSNLSW XLXXXXXW 60 95 19 ENV175 LLVLQAGF XLXXXXXF 61 95 19 ENV 175 LLVLQAGFF XLXXXXXXF 20.0121 62100 20 ENV 338 LLVPFVQW XLXXXXXW 63 100 20 ENV 338 LLVPFVQWF XLXXXXXXF64 85 17 NUC 100 LLWFHISCLTF XLXXXXXXXXF 65 95 19 NUC 45 LSFLPSDFXSXXXXXF 66 95 19 NUC 45 LSFLPSDFF XSXXXXXXF 20.0123 67 95 19 POL 415LSLDVSAAF XSXXXXXXF 68 95 19 POL 415 LSLDVSAAFY XSXXXXXXXY 2.0239 *4.2000 69 100 20 ENV 336 LSLLVPFVQW XSXXXXXXXW 70 100 20 ENV 336LSLLVPFVQWF XSXXXXXXXXF 71 95 19 X 53 LSLRGLPVCAF XSXXXXXXXXF 72 95 19POL 510 LSPFLLAQF XSXXXXXXF 73 75 15 ENV 349 LSPTVWLSVIW XSXXXXXXXXW 7485 17 POL 742 LSRKYTSF XSXXXXXF 75 85 17 POL 742 LSRKYTSFPW XSXXXXXXXW76 75 15 ENV 16 LSVPNPLGF XSXXXXXXF 77 75 15 NUC 137 LTFGRETVLEYXTXXXXXXXXY 78 90 18 ENV 189 LTIPQSLDSW XTXXXXXXXW 79 90 18 ENV 189LTIPQSLDSWW XTXXXXXXXXW 80 90 18 POL 404 LTNLLSSNLSW XTXXXXXXXXW 81 9519 ENV 176 LVLQAGFF XVXXXXXF 82 100 20 ENV 339 LVPFVQWF XVXXXXXF 83 10020 POL 377 LWDFSQF XVXXXXXF 84 85 17 ENV 360 MMWYWGPSLY XMXXXXXXXY1039.01 * 0.0810 85 75 15 X 103 MSTTDLEAY XSXXXXXXY 2.0126 * 0.8500 8675 15 X 103 MSTTDLEAYF XSXXXXXXXF 87 95 19 POL 42 NLGNLNVSIPWXLXXXXXXXXW 88 90 18 POL 406 NLLSSNLSW XLXXXXXXW 89 95 19 POL 45NLNVSIPW XLXXXXXW 90 75 15 ENV 15 NLSVPNPLGF XLXXXXXXXF 91 90 18 POL 738NSVVLSRKY XSXXXXXXY 2.0123 0.0005 92 100 20 ENV 380 PIFFCLWVY XIXXXXXXY1.0843 0.0078 93 100 20 ENV 314 PIPSSWAF XIXXXXXF 94 100 20 POL 124PLDKGIKPY XLXXXXXXY 1.0174 * 0.0190 95 100 20 POL 124 PLDKGIKPYYXLXXXXXXXY 1.0541 * 0.1600 96 100 20 ENV 377 PLLPIFFCLW XLXXXXXXXW 97 9519 ENV 174 PLLVLQAGF XLXXXXXXF 98 95 19 ENV 174 PLLVLQAGFF XLXXXXXXXF 9980 16 POL 505 PMGVGLSPF XMXXXXXXF 100 85 17 POL 797 PTTGRTSLY XTXXXXXXY1.0208 * 0.7700 101 75 15 ENV 351 PTVWLSVIW XTXXXXXXW 102 85 17 POL 612PVNRPIDW XVXXXXXW 103 95 19 POL 685 QVFADATPTG XVXXXXXXXXW 104 90 18 POL624 RIVGLLGF XIXXXXXF 105 75 15 POL 106 RLKLIMPARF XLXXXXXXXF 106 75 15POL 106 RLKLIMPARFY XLXXXXXXXXY 107 95 19 POL 376 RLVVDFSQF XLXXXXXXF20.0122 108 90 18 POL 353 RTPARVTGGVF XTXXXXXXXXF 109 100 20 POL 49SIPWTHKVGNF XIXXXXXXXXF 110 95 19 ENV 194 SLDSWWTSLNF XLXXXXXXXXF 111 9519 POL 416 SLDVSAAF XLXXXXXF 112 95 19 POL 416 SLDVSAAFY XLXXXXXXY1.0186 * 17.2000 113 100 20 ENV 337 SLLVPFVQW XLXXXXXXW 114 100 20 ENV337 SLLVPFVQWF XLXXXXXXXF 115 95 19 X 54 SLRGLPVCAF XLXXXXXXXF 20.0259116 90 18 X 64 SSAGPCALRF XSXXXXXXXF 26.0374 117 75 15 X 104 STTDLEAYXTXXXXXY 118 75 15 X 104 STTDLEAYF XTXXXXXXF 119 75 15 ENV 17 SVPNPLGFXVXXXXXF 120 90 18 POL 739 SVVLSRKY XVXXXXXY 26.0029 121 85 17 POL 739SVVLSRKYTSF XVXXXXXXXXF 122 90 18 ENV 190 TIPQSLDSW XIXXXXXXW 123 90 18ENV 190 TIPQSLDSWW XIXXXXXXXW 124 100 20 POL 150 TLWKAGILY XLXXXXXXY1.0177 * 0.0017 125 75 15 X 105 TTDLEAYF XTXXXXXF 126 85 17 POL 798TTGRTSLY XTXXXXXY 26.0030 127 80 16 NUC 16 TVQASKLCLGW XVXXXXXXXXW 12875 15 ENV 352 TVWLSVIW XVXXXXXW 129 85 17 POL 741 VLSRKYTSF XLXXXXXXF130 85 17 POL 741 VLSRKYTSFPW XLXXXXXXXXW 131 85 17 POL 740 VVLSRKYTSFXVXXXXXXXF 20.0261 132 80 16 POL 759 WILRGTSF XIXXXXXF 133 80 16 POL 759WILRGTSFVY XIXXXXXXXY 1.0572 0.0023 134 95 19 NUC 125 WIRTPPAY XIXXXXXY26.0031 135 80 16 POL 751 WLLGCAANW XLXXXXXXW 136 95 19 POL 414WLSLDVSAAF XLXXXXXXXF 137 95 19 POL 414 WLSLDVSAAFY XLXXXXXXXXY 26.0551138 100 20 ENV 335 WLSLLVPF XLXXXXXF 139 100 20 ENV 335 WLSLLVPFVQWXLXXXXXXXXW 140 85 17 NUC 26 WLWGMDIDPY XLXXXXXXXY 1.0774 * 0.0810 14195 19 ENV 237 WMCLRRFIIF XMXXXXXXXF 20.0266 142 85 17 ENV 359 WMMWYWGPSXMXXXXXXXXY 26.0552 * 143 100 20 POL 52 WTHKVGNF XTXXXXXF 144 100 20 POL122 YLPLDKGIKPY XLXXXXXXXXY 26.0553 145 90 18 NUC 118 YLVSFGVW XLXXXXXW146 80 16 POL 493 YSHPIILGF XSXXXXXXF 147 85 17 POL 580 YSLNFMGYXSXXXXXY 26.0032 148

TABLE VII HBV A02 SUPER MOTIF (With binding information) Conser- Fre- C-vancy quency Protein Position Sequence P2 term Peptide AA Filed A*0201A*0202 A*0203 A*0206 A*6802 SEQ ID NO: 85 17 POL 721 AACFARSRSGA A A 11149 85 17 POL 431 AAMPHLLV A V 8 150 80 16 POL 756 AANWILRGT A T 9 15195 19 POL 632 AAPFTQCGYPA A A 11 152 95 19 POL 521 AICSVVRRA I A 5.00259 0.0001 153 90 18 NUC 58 AILCWGEL I L 8 154 90 18 NUC 58 AILCWGELM I M9 155 95 19 POL 642 ALMPLYACI L I 927.15 9 * 0.5000 0.0340 3.3000 0.25000.0470 156 80 16 ENV 108 AMQWNSTT M T 8 157 75 15 X 102 AMSTTDLEA M A3.0051 9 0.0013 158 95 19 POL 690 ATPTGWGL T L 8 159 80 16 POL 690ATPTGWGLA T A 9 160 75 15 POL 690 ATPTGWGLAI T I 10 161 95 19 POL 397AVPNLQSL V L 8 162 95 19 POL 397 AVPNLQSLT V T 5.0026 9 0.0001 163 95 19POL 397 AVPNLQSLTNL V L 11 164 80 16 POL 755 CAANWILRGT A T 10 165 95 19X 61 CAFSSAGPCA A A 5.0090 10 0.0001 166 95 19 X 61 CAFSSAGPCAL A L 11167 90 18 X 69 CALRFTSA A A 8 168 100 20 ENV 312 CIPIPSSWA I A 5.0007 90.0010 169 80 16 ENV 312 CIPIPSSWAFA I A 11 170 90 18 POL 533 CLAFSYMDDVL V 1.0559 10 0.0008 171 90 18 POL 533 CLAFSYMDDW L V 11 172 85 17 NUC23 CLGWLWGM L M 8 173 85 17 NUC 23 CLGWLWGMDI L I 3.0210 10 0.0093 174100 20 ENV 253 CLIFLLVL L L Chisari 8 0.0002 175 4.011 100 20 ENV 253CLIFLLVLL L L 1.0836 9 0.0006 176 95 19 ENV 239 CLRRFIIFL L L 1.0829 90.0002 177 75 15 ENV 239 CLRRFIIFLFI L I Chisari 11 0.0004 178 4.055 9018 NUC 107 CLTFGRET L T 8 179 90 18 NUC 107 CLTFGRETV L V 1.0160 90.0001 180 100 20 ENV 310 CTCIPIPSSWA T A 11 181 95 19 POL 689 DATPTGWGLA L 5.0027 9 0.0001 182 80 16 POL 689 DATPTGWGLA A A 10 183 75 15 POL689 DATPTGWGLAI A I 11 184 90 18 NUC 31 DIDPYKEFGA I A 10 185 85 17 NUC29 DLLDTASA L A 8 186 85 17 NUC 29 DLLDTASAL L L 1.0154 9 0.0001 187 9519 POL 40 DLNLGNLNV L V 927.30 9 0.0004 188 95 19 POL 40 DLNLGNLNVSI L I11 189 80 16 NUC 32 DTASALYREA T A 10 190 80 16 NUC 32 DTASALYREAL T L11 191 95 19 X 14 DVLCLRPV V V 8 192 95 19 X 14 DVLCLRPVGA V A 5.0091 100.0001 193 90 18 POL 541 DVVLGAKSV V V 1.0190 9 0.0003 194 100 20 POL 17EAGPLEEEL A L 5.0028 9 0.0001 195 80 16 X 122 ELGEEFL L L 8 196 90 18POL 718 ELLAACFA L A 8 197 75 15 NUC 142 ETVLEYLV T V 8 198 95 19 POL687 FADATPTGWGL A L 11 199 85 17 POL 724 FARSRSGA A A 8 200 80 16 POL821 FASPLHVA A A 8 201 95 19 POL 396 FAVPNLQSL A L 9 202 95 19 POL 396FAVPNLQSLT A T 5.0083 10 0.0003 203 80 16 ENV 243 FIIFLFIL I L Chisari 80.0006 204 4.047 80 16 ENV 243 FIIFLFILL I L 1.0830 9 0.0002 205 80 16ENV 243 FIIFLFILLL I L 1.0894 10 0.0012 206 80 16 ENV 248 FILLLCLI I IChisari 8 0.0003 207 4.048 80 16 ENV 248 FILLLCLIFL I L 1.0895 10 *0.0280 208 80 16 ENV 248 FILLLCLIFLL I L Chisari 11 0.0010 209 4.049 8016 ENV 246 FLFILLLCL L L 1.0832 9 0.0002 210 80 16 ENV 246 FLFILLLCLI LI 3.0206 10 0.0013 211 75 15 ENV 171 FLGPLLVL L L 8 212 75 15 ENV 171FLGPLLVLQA L A 3.0205 10 * 0.0190 213 95 19 POL 513 FLLAQFTSA L A1069.07 9 * 0.2400 214 95 19 POL 513 FLLAQFTSAI L I 1147.13 10 * 0.21000.0320 7.0000 0.1100 0.0880 215 95 19 POL 562 FLLSLGIHL L L 927.11 9 *0.6500 0.0010 0.0100 0.1100 0.0035 216 80 16 ENV 183 FLLTRILT L T 8 21780 16 ENV 183 FLLTRILTI L I 777.03 9 * 0.5100 0.0430 8.0000 0.20000.0010 218 95 19 ENV 256 FLLVLLDYQGM L M 11 219 100 20 POL 363FLVDKNPHNT L T 5.0084 10 0.0012 220 95 19 POL 656 FTFSPTYKA T A 1147.159 * 0.0056 0.0150 0.0031 0.8000 7.3000 221 95 19 POL 656 FTFSPTYKAFL T L11 222 95 19 POL 59 FTGLYSST T T 8 223 90 18 POL 59 FTGLYSSTV T V20.0118 9 0.0005 224 95 19 POL 635 FTQCGYPA T A 8 225 95 19 POL 635FTQCGYPAL T L 5.0031 9 0.0009 226 95 19 POL 635 FTQCGYPALM T M 5.0085 100.0024 227 95 19 POL 518 FTSAICSV T V 8 228 95 19 POL 518 FTSAICSVV T V5.0032 9 0.0090 229 95 19 ENV 346 FVGLSPTV V V 8 230 95 19 ENV 346FVGLSPTVWL V L 1.0931 10 0.0008 231 90 18 X 132 FVLGGCRHKL V L Chisari10 0.0030 232 4.114 90 18 X 132 FVLGGCRHKLV V V 11 233 95 19 ENV 342FVQWFVGL V L 8 234 95 19 ENV 342 FVQWFVGLSPT V T 11 235 90 18 POL 766FVYVPSAL V L 8 236 90 18 POL 766 FVYVPSALNPA V A 11 237 95 19 X 50GAHLSLRGL A L 5.0040 9 0.0001 238 90 18 X 50 GAHLSLRGLPV A V 11 239 8517 POL 545 GAKSVQHL A L 8 240 85 17 POL 545 GAKSVQHLESL A L 11 241 75 15POL 567 GIHLNPNKT I T 9 242 90 18 POL 155 GILYKRET I T 8 243 90 18 POL155 GILYKRETT I T 9 244 85 17 POL 682 GLCQVFADA L A 1142.04 9 * 0.0024245 85 17 POL 682 GLCQVFADAT L T 10 246 95 19 POL 627 GLLGFAAPFT L T5.0086 10 0.0049 247 85 17 ENV 62 GLLGWSPQA L A 1142.07 9 * 0.40000.0003 0.0350 0.2800 0.0005 248 95 19 X 57 GLPVCAFSSA L A 5.0092 100.0008 249 95 19 POL 509 GLSPFLLA L A 8 250 95 19 POL 509 GLSPFLLAQFT LT 11 251 100 20 ENV 348 GLSPTVWL L L Chisari 8 0.0036 252 4.012 75 15ENV 348 GLSPTVWLSV L V 1.0518 10 * 0.2800 253 75 15 ENV 348 GLSPTVWLSVIL I Chisari 11 0.0036 254 4.031 90 18 ENV 265 GMLPVCPL M L 8 255 90 18POL 735 GTDNSVVL T L 8 256 75 15 ENV 13 GTNLSVPNPL T L 10 257 80 16 POL763 GTSFVYVPSA T A 10 258 80 16 POL 763 GTSFVYVPSAL T L 11 259 80 16 POL507 GVGLSPFL V L 8 260 80 16 POL 507 GVGLSPFLL V L Chisari 9 0.0002 2614.082 80 16 POL 507 GVGLSPFLLA V A 10 262 95 19 NUC 123 GVWIRTPPA V A3.0040 9 0.0030 263 90 18 NUC 104 HISCLTFGRET I T 11 264 80 16 POL 435HLLVGSSGL L L 927.43 9 0.0031 265 90 18 X 52 HLSLRGLPV L V 927.02 90.0014 266 90 18 X 52 HLSLRGLPVCA L A 11 267 80 16 POL 491 HLYSHPII L I17.0256 8 268 80 16 POL 491 HLYSHPIIL L L 927.47 9 * 0.2200 0.00030.9300 0.1700 0.0530 269 85 17 POL 715 HTAELLAA T A 8 270 85 17 POL 715HTAELLAACFA T A 11 271 100 20 NUC 52 HTALRQAI T I 8 272 95 19 NUC 52HTALRQAIL T L 5.0021 9 0.0001 273 100 20 POL 149 HTLWKAGI T I 8 274 10020 POL 149 HTLWKAGIL T L 5.0033 9 0.0001 275 80 16 ENV 244 IIFLFILL I LChisari 8 0.0004 276 4.051 80 16 ENV 244 IIFLFILLL I L 1.0831 9 0.0002277 80 16 ENV 244 IIFLFILLLCL I L Chisari 11 0.0002 278 4.052 80 16 POL497 IILGFRKI I I 8 279 80 16 POL 497 IILGFRKIPM I M 10 280 90 18 NUC 59ILCWGELM L M 8 281 80 16 POL 498 ILGFRKIPM L M 3.0016 9 0.0002 282 10020 ENV 249 ILLLCLIFL L L 1137.04 9 * 0.0015 283 100 20 ENV 249ILLLCLIFLL L L 1069.08 10 * 0.0190 0.0001 0.0002 0.1300 0.0015 284 10020 ENV 249 ILLLCLIFLLV L V Chisari 11 0.0056 285 4.013 80 16 POL 760ILRGTSFV L V 8 286 80 16 POL 760 ILRGTSFVYV L V 1.0573 10 * 0.0160 287100 20 NUC 139 ILSTLPET L T 8 288 100 20 NUC 139 ILSTLPETT L T 5.0022 90.0001 289 100 20 NUC 139 ILSTLPETTV L V 1069.14 10 * 0.0210 0.00850.0770 0.3100 0.0067 290 100 20 NUC 139 ILSTLPETTVV L V 11 291 95 19 ENV188 ILTIPQSL L L 8 292 90 18 POL 156 ILYKRETT L T 8 293 90 18 POL 625IVGLLGFA V A 8 294 90 18 POL 625 IVGLLGFAA V A 3.0041 9 0.0009 295 90 18POL 153 KAGILYKRET A T 10 296 90 18 POL 153 KAGILYKRETT A T 11 297 80 16POL 503 KIPMGVGL I L 8 298 85 17 NUC 21 KLCLGWLWGM L M 1142.02 10 *0.0001 299 95 19 POL 489 KLHLYSHPI L I 927.46 9 * 0.0690 0.0340 2.70000.5900 0.0015 300 80 16 POL 489 KLHLYSHPII L I 10 301 80 16 POL 489KLHLYSHPIIL L L 11 302 80 16 POL 610 KLPVNRPI L I 8 303 95 19 POL 574KTKRWGYSL T L 5.0034 9 0.0001 304 85 17 POL 620 KVCQRIVGL V L 1.0198 90.0003 305 85 17 POL 620 KVCQRIVGLL V L 1.0567 10 0.0001 306 95 19 POL55 KVGNFTGL V L 17.0116 8 307 85 17 X 91 KVLHKRTL V L 8 308 85 17 X 91KVLHKRTLGL V L Chisari 10 0.0004 309 4.115 90 18 POL 534 LAFSYMDDV A V20.0119 9 0.0002 310 90 18 POL 534 LAFSYMDDVV A V 20.0257 10 0.0003 31190 18 POL 534 LAFSYMDDVVL A L 11 312 95 19 POL 515 LAQFTSAI A I 8 313 9519 POL 515 LAQFTSAICSV A V 11 314 100 20 ENV 254 LIFLLVLL I L Chisari 80.0025 315 4.014 95 19 POL 514 LLAQFTSA L A 8 316 95 19 POL 514LLAQFTSAI L I 1069.05 9 * 0.1000 0.2700 3.7000 0.2600 0.7900 317 100 20ENV 251 LLCLIFLL L L Chisari 8 0.0004 318 4.015 100 20 ENV 251 LLCLIFLLVL V 1137.03 9 * 0.0048 319 100 20 ENV 251 LLCLIFLLVL L L 1.0898 100.0075 320 100 20 ENV 251 LLCLIFLLVLL L L Chisari 11 0.0013 321 4.016 8517 NUC 30 LLDTASAL L L 8 322 95 19 ENV 260 LLDYQGML L L Chisari 8 0.0004323 4.021 90 18 ENV 260 LLDYQGMLPV L V 1137.02 10 * 0.0980 0.0001 0.02000.6700 0.0009 324 80 16 POL 752 LLGCAANWI L I 927.22 9 0.0011 325 80 16POL 752 LLGCAANWIL L L 1.0912 10 * 0.0140 326 95 19 POL 628 LLGFAAPFT LT 5.0035 9 0.0008 327 85 17 ENV 63 LLGWSPQA L A 8 328 75 15 ENV 63LLGWSPQAQGI L I 11 329 100 20 ENV 250 LLLCLIFL L L Chisari 8 0.0006 3304.017 100 20 ENV 250 LLLCLIFLL L L 1090.05 9 * 0.0065 331 100 20 ENV 250LLLCLIFLLV L V 1137.01 10 * 0.0036 332 100 20 ENV 250 LLLCLIFLLVL L LChisaRi 11 0.0005 333 4.018 100 20 ENV 378 LLPIFFCL L L Chisari 8 0.0055334 4.019 100 20 ENV 378 LLPIFFCLWV L V 1069.10 10 * 0.0320 0.00080.0150 0.8000 0.0005 335 95 19 POL 563 LLSLGIHL L L 8 336 90 18 POL 407LLSSNLSWL L L 927.41 9 * 0.0110 0.0780 3.9000 0.2700 0.0100 337 90 18POL 407 LLSSNLSWLSL L L 11 338 80 16 ENV 184 LLTRILTI L I Chisari 80.0026 339 4.053 80 16 POL 436 LLVGSSGL L L 8 340 95 19 ENV 257LLVLLDYQGM L M 3.0207 10 0.0050 341 95 19 ENV 257 LLVLLDYQGML L L 11 34290 18 ENV 175 LLVLQAGFFL L L 1090.06 10 * 0.0310 0.0037 0.0045 0.15000.0110 343 90 18 ENV 175 LLVLQAGFFLL L L Chisari 11 0.0074 344 4.028 9519 ENV 338 LLVPFVQWFV L V 1069.06 10 * 0.6700 0.3800 1.7000 0.29000.1400 345 90 18 NUC 100 LLWFHISCL L L 1142.01 9 * 0.0130 0.0002 0.04200.3100 0.0098 346 85 17 NUC 100 LLWFHISCLT L T 10 347 95 19 POL 643LMPLYACI M I 17.0130 8 348 95 19 NUC 108 LTFGRETV T V 8 349 75 15 NUC137 LTFGRETVL T L 9 350 90 18 POL 404 LTNLLSSNL T L 9 351 80 16 ENV 185LTRILTIPQSL T L 11 352 85 17 POL 99 LTVNEKRRL T L 9 353 100 20 POL 364LVDKNPHNT V T 5.0036 9 0.0001 354 95 19 ENV 258 LVLLDYQGM V M 3.0034 90.0001 355 95 19 ENV 258 LVLLDYQGML V L 1.0515 10 0.0001 356 90 18 ENV176 LVLQAGFFL V L 1.0827 9 0.0096 357 90 18 ENV 176 LVLQAGFFLL V L1132.17 10 * 0.0022 358 90 18 ENV 176 LVLQAGFFLLT V T 11 359 95 19 ENV339 LVPFVQWFV V V 1132.01 9 * 0.0420 0.0150 0.0048 0.7900 2.8000 360 9519 ENV 339 LVPFVQWFVGL V L 11 361 90 18 NUC 119 LVSFGVWI V I Chisari 80.0004 362 4.078 90 18 NUC 119 LVSFGVWIRT V T 10 363 85 17 ENV 360MMWYWGPSL M L 1039.03 9 * 0.6400 364 100 20 NUC 136 NAPILSTL A L 8 365100 20 NUC 136 NAPILSTLPET A T 11 366 95 19 POL 42 NLGNLNVSI L I 3.00089 0.0047 367 90 18 POL 406 NLLSSNLSWL L L 1.0549 10 0.0016 368 95 19 POL45 NLNVSIPWT L T 5.0037 9 0.0005 369 100 20 POL 400 NLQSLTNL L L 8 370100 20 POL 400 NLQSLTNLL L L 927.40 9 0.0047 371 75 15 ENV 15 NLSVPNPL LL 8 372 90 18 POL 411 NLSWLSLDV L V 927.42 9 * 0.0650 0.0051 0.64000.1600 0.0990 373 90 18 POL 411 NLSWLSLDVSA L A 11 374 100 20 POL 47NVSIPWTHKV V V 1.0532 10 0.0001 375 100 20 POL 430 PAAMPHLL A L 8 376 8517 POL 430 PAAMPHLLV A V 9 377 90 18 POL 775 PADDPSRGRL A L 10 378 90 18ENV 131 PAGGSSSGT A T 9 379 90 18 ENV 131 PAGGSSSGTV A V 10 380 95 19POL 641 PALMPLYA A A 8 381 95 19 POL 641 PALMPLYACI A I 5.0087 10 0.0001382 75 15 X 145 PAPCNFFT A T 8 383 75 15 X 145 PAPCNFFTSA A A 10 384 8016 X 11 PARDVLCL A L 8 385 75 15 X 11 PARDVLCLRPV A V 11 386 90 18 POL355 PARVTGGV A V 8 387 90 18 POL 355 PARVTGGVFL A L 10 388 90 18 POL 355PARVTGGVFLV A V 11 389 95 19 NUC 130 PAYRPPNA A A 8 390 95 19 NUC 130PAYRPPNAPI A I 5.0081 10 0.0001 391 95 19 NUC 130 PAYRPPNAPIL A L 11 39285 17 POL 616 PIDWKVCQRI I I Chisari 10 0.0001 393 4.091 85 17 POL 616PIDWKVCQRIV I V 11 394 100 20 ENV 380 PIFFCLWV I V 8 395 100 20 ENV 380PIFFCLWVYI I I Chisari 10 0.0004 396 3.074 85 17 POL 713 PIHTAELL I L 8397 85 17 POL 713 PIHTAELLA I A 9 398 85 17 POL 713 PIHTAELLAA I A 10399 80 16 POL 496 PIILGFRKI I I 927.48 9 0.0001 400 80 16 POL 496PIILGFRKIPM I M 11 401 100 20 NUC 138 PILSTLPET I T 5.0023 9 0.0001 402100 20 NUC 138 PILSTLPETT I T 5.0082 10 0.0001 403 100 20 NUC 138PILSTLPETTV I V Chisari 11 0.0001 404 5.125 80 16 ENV 314 PIPSSWAFA I A9 405 95 19 POL 20 PLEEELPRL L L 927.29 9 0.0003 406 90 18 POL 20PLEEELPRLA L A 3.0225 10 0.0001 407 95 19 ENV 10 PLGFFPDHQL L L 1.051110 0.0002 408 100 20 POL 427 PLHPAAMPHL L L 1.0550 10 0.0001 409 100 20POL 427 PLHPAAMPHLL L L 11 410 100 20 ENV 377 PLLPIFFCL L L 1069.13 9 *0.0650 0.0001 0.0018 0.1100 0.0047 411 100 20 ENV 377 PLLPIFFCLWV L V 11412 90 18 ENV 174 PLLVLQAGFFL L L Chisari 11 0.0008 413 4.029 80 16 POL711 PLPIHTAEL L L 927.19 9 0.0004 414 80 16 POL 711 PLPIHTAELL L L1.0569 10 0.0001 415 80 16 POL 711 PLPIHTAELLA L A 11 416 75 15 POL 2PLSYQHFRKL L L 1.0527 10 0.0001 417 75 15 POL 2 PLSYQHFRKLL L L 11 41885 17 POL 98 PLTVNEKRRL L L 1.0536 10 0.0001 419 80 16 POL 505PMGVGLSPFL M L 1.0557 10 0.0001 420 80 16 POL 505 PMGVGLSPFLL M L 11 42175 15 POL 692 PTGWGLAI T I 8 422 80 16 ENV 219 PTSNHSPT T T 8 423 85 17POL 797 PTTGRTSL T L 8 424 85 17 POL 797 PTTGRTSLYA T A 10 425 80 16 NUC15 PTVQASKL T L 8 426 80 16 NUC 15 PTVQASKLCL T L 10 427 75 15 ENV 351PTVWLSVI T I 8 428 75 15 ENV 351 PTVWLSVIWM T M 10 429 95 19 X 59PVCAFSSA V A 8 430 85 17 POL 612 PVNRPIDWKV V V 1.0566 10 0.0002 431 9519 POL 654 QAFTFSPT A T 8 432 95 19 POL 654 QAFTFSPTYKA A A 11 433 95 19ENV 179 QAGFFLLT A T 8 434 80 16 ENV 179 QAGFFLLTRI A I 10 435 80 16 ENV179 QAGFFLLTRIL A L 11 436 90 18 NUC 57 QAILCWGEL A L 9 437 90 18 NUC 57QAILCWGELM A M 10 438 95 19 ENV 107 QAMQWNST A T 8 439 80 16 ENV 107QAMQWNSTT A T 9 440 80 16 NUC 18 QASKLCLGWL A L 10 441 80 16 X 8QLDPARDV L V Chisari 8 0.0001 442 4.116 80 16 X 8 QLDPARDVL L L 927.01 90.0001 443 80 16 X 8 QLDPARDVLCL L L Chisari 11 0.0001 444 4.073 90 18NUC 99 QLLWFHISCL L L 1142.03 10 * 0.0060 445 85 17 NUC 99 QLLWFHISCLT LT 11 446 95 19 POL 685 QVFADATPT V T 5.0038 9 0.0001 447 95 19 POL 528RAFPHCLA A A 8 448 80 16 ENV 187 RILTIPQSL I L Chisari 9 0.0010 4494.054 90 16 POL 624 RIVGLLGFA I A 9 450 90 18 POL 624 RIVGLLGFAA I A 10451 75 15 POL 106 RLKLIMPA L A 8 452 90 18 POL 353 RTPARVTGGV T V 10 45395 19 NUC 127 RTPPAYRPPNA T A 11 454 95 19 POL 36 RVAEDLNL V L 8 455 9018 POL 36 RVAEDLNLGNL V L 11 456 80 16 POL 818 RVHFASPL V L 8 457 75 15POL 818 RVHFASPLHV V V 1.0576 10 0.0001 458 75 15 POL 818 RVHFASPLHVA VA 11 459 100 20 POL 357 RVTGGVFL V L 8 460 100 20 POL 357 RVTGGVFLV V V1.0181 9 0.0041 461 90 18 X 65 SAGPCALRFT A T 10 462 95 19 POL 520SAICSVVRRA A A 5.0088 10 0.0001 463 90 18 NUC 35 SALYREAL A L 8 464 10020 POL 49 SIPWTHKV I V 8 465 95 19 ENV 194 SLDSWWTSL L L F126.64 9 46675 15 POL 565 SLGIHLNPNKT L T 11 467 95 19 ENV 337 SLLVPFVQWFV L V 11468 75 15 POL 581 SLNFMGYV L V 8 469 75 15 POL 581 SLNFMGYVI L I 927.129 0.0038 470 95 19 X 54 SLRGLPVCA L A 3.0030 9 0.0007 471 90 18 POL 403SLTNLLSSNL L L 1.0548 10 0.0014 472 75 15 ENV 280 STGPCKTCT T T 9 473100 20 NUC 141 STLPETTV T V 8 474 100 20 NUC 141 STLPETTVV T V 5.0024 90.0019 475 80 16 ENV 85 STNRQSGRQPT T T 11 476 85 17 POL 548 SVQHLESL VL 8 477 80 16 ENV 330 SVRFSWLSL V L Chisari 9 0.0001 478 4.025 80 16 ENV330 SVRFSWLSLL V L Chisari 10 0.0004 479 4.026 80 16 ENV 330 SVRFSWLSLLVV V 11 480 90 18 POL 739 SVVLSRKYT V T 9 481 95 19 POL 524 SVVRRAFPHCL VL 11 482 85 17 POL 716 TAELLAACFA A A 10 483 95 19 NUC 53 TALRQAIL A L 8484 80 16 NUC 33 TASALYREA A A 9 485 80 16 NUC 33 TASALYREAL A L 10 48690 18 ENV 190 TIPQSLDSWWT I T 11 487 100 20 NUC 142 TLPETTVV L V 8 488100 20 POL 150 TLWKAGIL L L 8 489 85 17 POL 798 TTGRTSLYA T A 9 490 7515 ENV 278 TTSTGPCKT T T 9 491 75 15 ENV 278 TTSTGPCKTCT T T 11 492 8517 POL 100 TVNEKRRL V L 8 493 80 18 NUC 16 TVQASKLCL V L 1.0365 9 0.0002494 75 15 ENV 352 TVWLSVIWM V M 3.0035 9 0.0002 495 95 19 POL 37VAEDLNLGNL A L 5.0089 10 0.0001 496 95 19 X 15 VLCLRPVGA L A 3.0028 90.0014 497 85 17 POL 543 VLGAKSVCHL L L 1.0560 10 0.0001 498 90 18 X 133VLGGCRHKL L L 927.08 9 0.0009 499 90 18 X 133 VLGGCRHKLV L V 1.0589 100.0001 500 85 17 X 92 VLHKRTLGL L L 927.03 9 0.0012 501 95 19 ENV 259VLLDYQGM L M 17.0107 8 502 95 19 ENV 259 VLLDYQGML L L 1069.09 9 *0.0440 0.0001 0.0210 0.9000 0.0002 503 90 18 ENV 259 VLLDYQGMLPV L V1147.14 11 * 0.5800 0.2200 4.9000 0.3400 0.0170 504 95 19 ENV 177VLQAGFFL L L Chisari 8 0.0019 505 4.027 95 19 ENV 177 VLQAGFFLL L L1013.14 9 * 0.0660 506 95 19 ENV 177 VLQAGFFLLT L T 5.0066 10 0.0011 507100 20 POL 358 VTGGVFLV T V 8 508 90 18 POL 542 VVLGAKSV V V 8 509 80 16POL 542 VVLGAKSVQHL V L 11 510 90 18 POL 740 VVLSRKYT V T 8 511 95 19POL 525 VVRRAFPHCL V L 2.0217 10 0.0003 512 95 19 POL 525 VVRRAFPHCLA VA 11 513 80 16 POL 759 WILRGTSFV I V 927.24 9 * 0.0270 514 80 16 POL 759WILRGTSFVYV I V 11 515 80 16 POL 751 WLLGCAANWI L I Chisari 10 0.0053516 4.101 80 16 POL 751 WLLGCAANWIL L L 11 517 100 20 POL 414 WLSLDVSA LA 8 518 95 19 POL 414 WLSLDVSAA L A 3.0023 9 0.0059 519 100 20 ENV 335WLSLLVPFV L V 1013.0102 9 * 1.1000 0.0380 7.2000 0.3600 0.0310 520 95 19ENV 237 WMCLRRFI M I 8 521 95 19 ENV 237 WMCLRRFII M I 1147.10 9 *0.0005 522 95 19 ENV 237 WMCLRRFIIFL M L Chisari 11 0.0019 523 4.024 8517 ENV 359 WMMWYWGPSL M L 1137.05 10 * 0.0009 524 100 20 POL 52WTHKVGNFT T T 5.0039 9 0.0001 525 95 19 POL 52 WTHKVGNFTGL T L 11 526100 20 POL 147 YLHTLWKA L A 8 527 100 20 POL 147 YLHTLWKAGI L I 1069.1110 * 0.0160 0.0005 0.5600 0.1000 0.0320 528 100 20 POL 147 YLHTLWKAGIL LL 11 529 100 20 POL 122 YLPLDKGI L I 8 530 90 18 NUC 118 YLVSFGVWI L I1090.12 9 * 0.3800 531 90 18 NUC 118 YLVSFGVWIRT L T 11 532 90 18 POL538 YMDDVVLGA M A 1090.14 9 * 0.0250 0.0001 0.0024 0.1000 0.0002 533 8517 POL 746 YTSFPWLL T L 8 534 75 15 POL 746 YTSFPWLLGCA T A 11 535 90 18POL 768 YVPSALNPA V A 3.0042 9 0.0039 536 388

TABLE VIII HBV A03 SUPER MOTIF (With binding information) C- ConservancyFrequency Protein Position Sequence P2 term Peptide AA Filed A*0301A*1101 A*3101 A*3301 A*6801 SEQ ID NO: 85 17 POL 721 AACFARSR A R26.0003 8 0.0004 0.0003 0.0056 0.0035 0.0014 537 95 19 POL 521 AICSVVRRI R 26.0004 8 −0.0002 0.0003 0.0014 −0.0009 0.0006 538 90 18 POL 772ALNPADDPSR L R 1.1090 10 0.0003 0.0001 539 85 17 X 70 ALRFTSAR L R26.0005 8 0.0047 0.0009 0.0450 0.0230 0.0004 540 80 16 POL 822 ASPLHVAWRS R 9 541 75 15 ENV 84 ASTNRQSGR S R 1150.60 9 0.0009 0.0002 0.00880.0008 0.0001 542 80 16 POL 755 CAANWILR A R 8 543 85 17 X 69 CALRFTSARA R 26.0149 9 * 0.0034 0.0230 1.5000 8.0000 0.7300 544 90 16 X 17CLRPVGAESR L R 1.1093 10 0.0011 0.0001 545 100 20 NUC 48 CSPHHTALR S R5.0055 9 * 0.0029 0.0001 0.0520 0.0250 0.0440 546 85 17 NUC 29DLLDTASALYR L R 26.0530 11 0.0042 −0.0003 −0.0012 3.7000 0.0410 547 8517 NUC 32 DTASALYR T R 26.0006 8 0.0004 −0.0002 −0.0009 0.0018 0.0009548 95 19 POL 17 EAGPLEEELPR A R 26.0531 11 −0.0009 −0.0003 −0.00120.0015 0.0110 549 90 18 POL 718 ELLAACFAR L R 1.0988 9 0.0002 0.0004 55085 17 POL 718 ELLAACFARSR L R 26.0532 11 0.0062 0.0016 0.0200 0.20000.1600 551 95 19 NUC 174 ETTVVRRR T R 26.0007 8 0.0003 −0.0002 −0.00090.1400 0.0027 552 80 16 NUC 174 ETTVVRRRGR T R 1.1073 10 0.0003 0.0001553 80 16 POL 821 FASPLHVAWR A R 10 554 90 18 X 83 FSSAGPCALR S R 10 55595 19 POL 856 FTFSPTYK T K 1147.19 8 * 0.0100 0.0100 0.0023 0.21000.0590 556 95 19 POL 518 FTSAICSVVR T R 1.1085 10 0.0003 0.0003 557 9519 POL 518 FTSAICSVVRR T R 26.0533 11 0.0065 0.0092 0.0170 0.0350 1.5000558 90 18 X 132 FVLGGCRHK V K 1090.03 9 * 0.0430 0.0090 559 75 15 POL567 GIHLNPNK I K 8 560 75 15 POL 567 GIHLNPNKTK I K 1.0563 10 0.00250.0011 0.0009 0.0009 0.0003 561 75 15 POL 567 GIHLNPNKTKR I R 11 562 8517 NUC 29 GMDIDPYK M K 26.0009 8 0.0006 0.0004 −0.0009 −0.0009 0.0001563 90 16 POL 735 GTDNSVVLSR T R 1090.04 10 * 0.0010 0.0420 0.00300.0019 0.0008 564 90 16 POL 735 GTDNSVVLSRK T K 1147.17 11 * 0.01400.5600 −0.0002 −0.0006 0.0001 565 95 19 NUC 123 GVWIRTPPAYR V R 26.053511 * 0.1900 0.1700 6.8000 0.7300 0.6600 566 90 18 NUC 104 HISCLTFGR I R1069.16 9 * 0.0160 0.0065 567 75 15 POL 569 HLNPNKTK L K 8 568 75 15 POL569 HLNPNKTKR L R 1.0983 9 0.0025 0.0001 569 100 20 POL 149 HTLWKAGILYKT K 1147.16 11 * 0.5400 0.4400 0.0370 0.0720 0.1900 570 90 18 NUC 105ISCLTFGR S R 26.0010 8 0.0004 0.0002 0.0017 −0.0009 0.0017 571 100 20POL 153 KAGILYKR A R 26.0011 8 0.0002 −0.0002 0.0015 −0.0009 0.0001 57280 16 POL 610 KLPVNRPIDWK L K 11 573 75 15 X 130 KVFVLGGCR V R 1.09939 * 0.0420 0.0820 0.6000 0.0710 0.0030 574 85 17 POL 720 LAACFARSR A R20.0129 9 0.0058 0.0065 575 90 18 POL 719 LLAACFAR L R 26.0012 8 0.00240.0003 0.0015 0.0029 0.0064 576 85 17 POL 719 LLAACFARSR L R 10 577 8517 NUC 30 LLDTASALYR L R 1.1070 10 0.0050 0.0002 578 80 16 POL 752LLGCAANWILR L R 11 579 75 15 POL 564 LSLGIHLNPNK S K 11 580 95 19 NUC169 LSTLPETTVVR S R 26.0537 11 −0.0009 0.0008 −0.0012 −0.0023 0.0078 58175 15 POL 3 LSYQHFRK S K 8 582 85 17 POL 99 LTVNEKRR T R 26.0013 8−0.0002 −0.0002 −0.0009 −0.0009 0.0001 583 90 18 NUC 119 LVSFGVWIR V R1090.08 9 * 0.0028 0.0120 584 100 20 POL 377 LVVDFSQFSR V R 1069.20 10 *0.0016 0.3600 0.0260 0.2300 0.4900 585 75 15 X 103 MSTTDLEAYFK S K 11586 90 18 NUC 75 NLEDPASR L R 26.0014 8 −0.0002 −0.0002 −0.0009 −0.00090.0001 587 95 19 POL 45 NLNVSIPWTHK L K 26.0538 11 −0.0009 0.0005−0.0012 −0.0023 0.0019 588 90 18 POL 738 NSVVLSRK S K 26.0015 8 0.00060.0010 −0.0009 −0.0009 0.0007 589 100 20 POL 47 NVSIPWTHK V K 1069.169 * 0.0620 0.0570 0.0002 0.0100 0.0320 590 90 18 POL 775 PADDPSRGR A R1150.35 9 0.0008 0.0002 0.0004 0.0015 0.0002 591 80 16 X 11 PARDVLCLR AR 1150.36 9 0.0002 0.0002 0.0100 0.0180 0.0002 592 75 15 ENV 83PASTNRQSGR A R 10 593 90 18 POL 616 PIDWKVCQR I R 1.0985 9 0.0002 0.0005594 80 16 POL 496 PIILGFRK I K 8 595 95 19 POL 20 PLEEELPR L R 26.0016 80.0002 −0.0002 −0.0009 −0.0009 0.0001 596 100 20 POL 2 PLSYQHFR L R26.0017 8 −0.0002 −0.0002 −0.0009 −0.0009 0.0001 597 75 15 POL 2PLSYQHFRK L K 1.0161 9 0.0011 0.0031 0.0006 0.0008 0.0002 598 85 17 POL98 PLTVNEKR L R 26.0018 8 0.0002 −0.0002 −0.0009 −0.0009 0.0001 599 8517 POL 98 PLTVNEKRR L R 1.0974 9 0.0008 0.0005 0.0004 0.0027 0.0002 60090 18 X 20 PVGAESRGR V R 1.0990 9 0.0002 0.0005 0.0004 0.0043 0.0002 60185 17 POL 612 PVNRPIDVVK V K 1142.06 9 * 0.0310 0.1400 0.0002 0.00060.0009 602 95 19 POL 654 QAFTFSPTYK A K 1090.10 10 * 0.0450 0.54000.0010 0.0057 1.2000 603 80 16 ENV 179 QAGFFLLTR A R 9 604 75 15 NUC 169QSPRRRRSQSR S R 28.0839 11 605 80 16 POL 189 QSSGILSR S R 8 606 75 15POL 106 RLKLIMPAR L R 1.0975 9 * 0.0950 0.0002 3.1000 0.0490 0.0002 60775 15 X 128 RLKVFVLGGCR L R 11 608 95 19 POL 376 RLVVDFSQFSR L R 26.053911 * 0.2800 3.8000 2.6000 1.2000 6.1000 609 95 19 NUC 183 RSPRRRTPSPR SR 26.0540 11 −0.0007 −0.0003 0.0190 −0.0023 0.0003 610 75 15 NUC 167RSQSPRRR S R 8 611 75 15 NUC 167 RSQSPRRRR S R 9 612 95 19 NUC 188RTPSPRRR T R 26.0019 8 −0.0002 −0.0002 0.0033 0.0014 0.0002 613 95 19NUC 188 RTPSPRRRR T R 1.0971 9 * 0.0054 0.0005 0.2000 0.0016 0.0003 614100 20 POL 357 RVTGGVFLVDK V K 1147.18 11 * 0.0190 0.0290 −0.0002−0.0003 0.0001 615 90 18 X 65 SAGPCALR A R 26.0020 8 −0.0002 0.00200.0029 0.0024 0.0360 616 95 19 POL 520 SAICSVVR A R 26.0021 8 −0.00020.0071 0.0280 0.0081 0.0690 617 95 19 POL 520 SAICSVVRR A R 1090.11 9 *0.0058 0.2100 0.0150 0.0650 0.3800 618 90 18 POL 771 SALNPADDPSR A R26.0542 11 −0.0004 −0.0003 −0.0012 −0.0023 0.0003 619 75 15 POL 565SLGIHLNPNK L K 28.0758 10 * 620 90 18 X 64 SSAGPCALR S R 26.0153 9 *0.0080 0.1400 0.3300 0.1600 0.7500 621 95 19 NUC 170 STLPETTVVR T R1069.21 10 * 0.0007 0.0600 0.0080 0.0240 0.0250 622 95 19 NUC 170STLPETTVVRR T R 1083.01 11 0.0150 1.4000 0.1000 0.1600 0.3100 623 80 16ENV 85 STNRQSGR T R 8 624 75 15 X 104 STTDLEAYFK T K 1.0584 10 * 0.00662.7000 625 85 17 POL 716 TAELLAACFAR A R 26.0544 11 0.0006 0.0023 0.00660.1600 0.0590 626 95 19 NUC 171 TLPETTVVR L R 1.0969 9 0.0008 0.00020.0009 0.0024 0.0180 627 95 19 NUC 171 TLPETTVVRR L R 1069.22 10 *0.0007 0.0230 0.0006 0.0120 0.0440 628 95 19 NUC 171 TLPETTVVRRR L R26.0545 11 * 0.0005 0.0160 0.0061 0.0710 0.6400 629 100 20 POL 150TLWKAGILYK L K 1069.15 10 * 5.3000 0.3600 0.0051 0.0010 0.0130 630 10020 POL 150 TLWKAGILYKR L R 26.0546 11 0.0082 0.0095 0.1000 0.1100 0.0640631 95 19 POL 519 TSAICSVVR S R 5.0057 9 0.0005 0.0008 0.0600 0.02000.0820 632 95 19 POL 519 TSAICSVVRR S R 1142.08 10 * 0.0018 0.00060.0030 0.0066 0.0048 633 75 15 X 105 TTDLEAYFK T K 1.0215 9 * 0.00060.9200 0.0006 0.0012 0.0170 634 75 15 ENV 278 TTSTGPCK T K 8 635 80 16NUC 175 TTVVRRRGR T R 1.0970 9 0.0008 0.0005 0.2500 0.1400 0.0095 636 8016 NUC 176 TVVRRRGR V R 3.0324 8 0.0003 0.0001 637 80 18 NUC 176TVVRRRGRSPR V R 28.0837 11 638 90 18 X 133 VLGGCRHK L K 26.0022 8 0.01500.0002 −0.0005 −0.0009 0.0001 639 80 16 ENV 177 VLQAGFFLLTR L R 11 64090 18 NUC 120 VSFGVWIR S R 26.0023 8 * 0.0040 0.0290 0.0750 0.02700.0360 641 100 20 POL 48 VSIPWTHK S K 26.0024 8 * 0.0130 0.0170 0.00310.0013 0.0004 642 100 20 POL 358 VTGGVFLVDK T K 1069.17 10 * 0.03900.0920 0.0002 0.0006 0.0022 643 100 20 POL 378 VVDFSQFSR V R 1069.19 9 *0.0015 0.0750 0.0013 0.0170 0.0330 644 80 16 NUC 177 VVRRRGRSPR V R1.1074 10 0.0027 0.0001 645 80 16 NUC 177 VVRRRGRSPRR V R 28.0838 11 64695 19 NUC 125 WIRTPPAYR I R 1.0968 9 0.0008 0.0005 647 90 18 POL 314WLQFRNSK L K 26.0025 8 −0.0002 0.0005 0.0020 0.0052 0.0001 648 85 17 NUC26 WLWGMDIDPYK L K 26.0547 11 0.0030 0.0013 −0.0003 0.0039 0.0490 649100 20 POL 122 YLPLDKGIK L K 1.0173 9 0.0001 0.0001 0.0006 0.0008 0.0002650 90 18 NUC 118 YLVSFGVWIR L R 1090.13 10 * 0.0005 0.0002 651 90 18POL 538 YMDDVVLGAK M K 1090.15 10 * 0.0330 0.0043 0.0002 0.0008 0.0001652 80 16 POL 493 YSHPIILGFR S R 10 653 80 16 POL 493 YSHPIILGFRK S K 11654 118

TABLE IX HBV A24 SUPER MOTIF (With binding information) Conservancy FreqProtein Position Sequence String Peptide Filed A*2401 SEQ ID NO: 95 19POL 529 AFPHCLAF XFXXXXXF 655 95 19 POL 529 AFPHCLAFSY XFXXXXXXXY 656 9519 POL 529 AFPHCLAFSYM XFXXXXXXXXM 657 95 19 X 62 AFSSAGPCAL XFXXXXXXXL5.0118 0.0012 658 90 18 POL 535 AFSYMDDVVL XFXXXXXXXL 13.0130 0.0009 65995 19 POL 655 AFTFSPTY XFXXXXXY 660 95 19 POL 655 AFTFSPTYKAFXFXXXXXXXXF 661 95 19 POL 521 AICSVVRRAF XIXXXXXXXF 662 90 18 NUC 58AILCWGEL XIXXXXXL 663 90 18 NUC 58 AILCWGELM XIXXXXXXM 664 95 19 POL 642ALMPLYACI XLXXXXXXI 3.0012 * 665 95 19 NUC 54 ALRQAILCW XLXXXXXXW 666 8016 ENV 108 AMQWNSTTF XMXXXXXXF 667 95 19 POL 690 ATPTGWGL XTXXXXXL 66875 15 POL 690 ATPTGWGLAI XTXXXXXXXI 669 95 19 POL 397 AVPNLQSL XVXXXXXL670 95 19 POL 397 AVPNLQSLTNL XVXXXXXXXXL 671 100 20 NUC 131 AYRPPNAPIXYXXXXXXI 5.0062 * 0.0260 672 100 20 NUC 131 AYRPPNAPIL XYXXXXXXXL2.0172 * 0.0220 673 75 15 POL 607 CFRKLPVNRPI XFXXXXXXXXI 674 100 20 ENV312 CIPIPSSW XIXXXXXW 675 100 20 ENV 312 CIPIPSSWAF XIXXXXXXXF 676 85 17NUC 23 CLGWLWGM XLXXXXXM 677 85 17 NUC 23 CLGWLWGMDI XLXXXXXXXI 2.0229678 100 20 ENV 253 CLIFLLVL XLXXXXXL 17.0248 679 100 20 ENV 253CLIFLLVLL XLXXXXXXL 1.0836 680 95 19 ENV 253 CLIFLLVLLDY XLXXXXXXXXY26.0548 681 95 19 ENV 239 CLRRFIIF XLXXXXXF 682 95 19 ENV 239 CLRRFIIFLXLXXXXXXL 1.0829 683 75 15 ENV 239 CLRRFIIFLF XLXXXXXXXF 684 75 15 ENV239 CLRRFIIFLFI XLXXXXXXXXI Chisari 685 4.055 100 20 ENV 310 CTCIPIPSSWXTXXXXXXXW 686 90 18 NUC 31 DIDPYKEF XIXXXXXF 687 85 17 NUC 29 DLLDTASALXLXXXXXXL 1.0154 688 85 17 NUC 29 DLLDTASALY XLXXXXXXXY 1.0519 * 689 9519 POL 40 DLNLGNLNVSI XLXXXXXXXXI 690 80 16 NUC 32 DTASALYREALXTXXXXXXXXL 691 85 17 POL 618 DWKVCQRI XWXXXXXI 692 85 17 POL 618DWKVCQRIVGL XWXXXXXXXXL 693 90 18 ENV 262 DYQGMLPVCPL XYXXXXXXXXL 3.04410.0002 694 80 16 X 122 ELGEEIRL XLXXXXXL 695 95 19 NUC 43 ELLSFLPSDFXLXXXXXXXF 696 95 19 NUC 43 ELLSFLPSDFF XLXXXXXXXXF 697 90 18 NUC 117EYLVSFGVW XYXXXXXXW 26.0150 698 90 18 NUC 117 EYLVSFGVWI XYXXXXXXXI13.0129 * 0.0340 699 100 20 ENV 382 FFCLWVYI XFXXXXXI 700 80 16 ENV 182FFLLTRIL XFXXXXXL 701 80 16 ENV 182 FFLLTRILTI XFXXXXXXXI 702 85 17 ENV13 FFPDHQLDPAF XFXXXXXXXXF 703 80 16 ENV 243 FIIFLFIL XIXXXXXL 17.0246704 80 16 ENV 243 FIIFLFILL XIXXXXXXL 1.0830 705 80 16 ENV 243FIIFLFILLL XIXXXXXXXL 1.0894 706 80 16 ENV 248 FILLLCLI XIXXXXXI Chisari707 4.048 80 16 ENV 248 FILLLCLIF XIXXXXXXF 708 80 16 ENV 248 FILLLCLIFLXIXXXXXXXL 1.0895 709 80 16 ENV 248 FILLLCLIFLL XIXXXXXXXXL Chisari 7104.049 80 16 ENV 246 FLFILLLCL XLXXXXXXL 1.0832 711 80 16 ENV 246FLFILLLCLI XLXXXXXXXI 3.0206 712 80 16 ENV 246 FLFILLLCLIF XLXXXXXXXXF713 75 15 ENV 171 FLGPLLVL XLXXXXXL 714 95 19 POL 513 FLLAQFTSAIXLXXXXXXXI 1147.13 * 715 95 19 POL 562 FLLSLGIHL XLXXXXXXL 1.0851 * 71680 16 ENV 183 FLLTRILTI XLXXXXXXI 3.0005 * 717 95 19 ENV 256 FLLVLLDYXLXXXXXY 26.0027 718 95 19 ENV 256 FLLVLLDYQGM XLXXXXXXXXM 719 95 19 POL656 FTFSPTYKAF XTXXXXXXXF 20.0262 720 95 19 POL 656 FTFSPTYKAFLXTXXXXXXXXL 721 95 19 POL 635 FTQCGYPAL XTXXXXXXL 5.0031 722 95 19 POL635 FTQCGYPALM XTXXXXXXXM 5.0085 723 95 19 ENV 346 FVGLSPTVW XVXXXXXXXW724 95 19 ENV 346 FVGLSPTVWL XVXXXXXXXL 1.0931 725 90 18 X 132FVLGGCRHKL XVXXXXXXXL 1.0588 726 95 19 ENV 342 FVQWFVGL XVXXXXXL17.0109 * 727 90 18 POL 766 FVYVPSAL XVXXXXXL 17.0260 * 728 95 19 POL630 GFAAPFTQCGY XFXXXXXXXXY 729 80 16 ENV 181 GFFLLTRI XFXXXXXI 730 8016 ENV 181 GFFLLTRIL XFXXXXXXL 731 80 16 ENV 181 GFFLLTRILTI XFXXXXXXXXI732 95 19 ENV 12 GFFPDHQL XFXXXXXL 733 75 15 ENV 170 GFLGPLLVL XFXXXXXXL734 80 16 POL 500 GFRKIPMGVGL XFXXXXXXXXL 735 95 19 POL 627 GLLGFAAPFXLXXXXXXF 20.0124 736 95 19 POL 509 GLSPFLLPAQF XLXXXXXXXF 737 100 20ENV 348 GLSPTVWL XLXXXXXL Chisari 738 4.012 75 15 ENV 348 GLSPTVWLSVIXLXXXXXXXXI Chisari 739 4.031 85 17 NUC 29 GMDIDPYKEF XMXXXXXXXF 26.0372740 90 18 ENV 265 GMLPVCPL XMXXXXXL 741 90 18 POL 735 GTDNSVVL XTXXXXXL742 75 15 ENV 13 GTNLSVPNPL XTXXXXXXXL 743 80 16 POL 763 GTSFVYVPSALXTXXXXXXXXL 744 80 16 POL 507 GVGLSPFL XVXXXXXL 745 80 16 POL 507GVGLSPFLL XVXXXXXXL 1.0850 746 95 19 NUC 123 GVWIRTPPAY XVXXXXXXXV1.0525 747 85 17 NUC 25 GWLWGMDI XWXXXXXI 748 85 17 NUC 25 GWLWGMDIDPYXWXXXXXXXXY 749 85 17 ENV 65 GWSPQAQGI XWXXXXXXI 20.0134 0.0024 750 8517 ENV 65 GWSPQAQGIL XWXXXXXXXL 20.0268 0.0003 751 95 19 POL 639GYPALMPL XYXXXXXL 752 95 19 POL 639 GYPALMPLY XYXXXXXXY 2.0060 * 0.0490753 95 19 ENV 234 GYRWMCLRRF XYXXXXXXXF 2.0171 * 0.0110 754 95 19 ENV234 GYRWMCLRRFI XYXXXXXXXXI 755 85 17 POL 579 GYSLNFMGY XYXXXXXXY 2.00580.0002 756 75 15 POL 579 GYSLNFMGYVI XYXXXXXXXXI 757 80 16 POL 820HFASPLHVAW XFXXXXXXXW 758 75 15 POL 7 HFRKLLLL XFXXXXXL 759 80 16 POL435 HLLVGSSGL XLXXXXXXL 1.0187 760 75 15 POL 569 HLNPNKTKRW XLXXXXXXXW761 80 16 POL 491 HLYSHPII XLXXXXXI 17.0256 762 80 16 POL 491 HLYSHPIILXLXXXXXXL 1.0849 * 763 80 16 POL 491 HLYSHPIILGF XLXXXXXXXXF 764 85 17POL 715 HTAELLAACF XTXXXXXXXF 765 100 20 NUC 52 HTALRQAI XTXXXXXI 766 9519 NUC 52 HTALRQAIL XTXXXXXXL 5.0021 767 95 19 NUC 52 HTALRQAILCWXTXXXXXXXXW 768 100 20 POL 149 HTLWKAGI XTXXXXXI 769 100 20 POL 149HTLWKAGIL XTXXXXXXL 5.0033 770 100 20 POL 149 HTLWKAGILY XTXXXXXXXY1.0542 * 771 100 20 POL 146 HYLHTLWKAGI XYXXXXXXXXI 772 100 20 ENV 381IFFCLWVY XFXXXXXY 773 100 20 ENV 381 IFFCLWVYI XFXXXXXXI 5.0058 0.0087774 80 16 ENV 245 IFLFILLL XFXXXXXL 775 80 16 ENV 245 IFLFILLLCLXFXXXXXXXL 776 80 16 ENV 245 IFLFILLLCLI XFXXXXXXXXI 777 95 19 ENV 255IFLLVLLDY XFXXXXXXY 778 80 16 ENV 244 IIFLFILL XIXXXXXL 17.0105 779 8016 ENV 244 IIFLFILLL XIXXXXXXL 1.0831 780 80 16 ENV 244 IIFLFILLLCLXIXXXXXXXXL Chisari 781 4.052 80 16 POL 497 IILGFRKI XIXXXXXI 17.0124 *782 80 16 POL 497 IILGFRKIPM XIXXXXXXXM 783 90 18 NUC 59 ILCWGELMXLXXXXXM 784 80 16 POL 498 ILGFRKIPM XLXXXXXXM 3.0016 785 100 20 ENV 249ILLLCLIF XLXXXXXF 786 100 20 ENV 249 ILLLCLIFL XLXXXXXXL 1.0833 * 787100 20 ENV 249 ILLLCLIFLL XLXXXXXXXL 1.0896 * 788 80 16 POL 760ILRGTSFVY XLXXXXXXY 1.0205 * 789 95 19 ENV 188 ILTIPQSL XLXXXXXL 790 9018 ENV 188 ILTIPQSLDSW XLXXXXXXXXW 791 90 18 POL 625 IVGLLGFAAPFXVXXXXXXXXF 792 85 17 ENV 358 IWMMWYWGPS XWXXXXXXXXL 1039.07 0.0004 79395 19 POL 395 KFAVPNLQSL XFXXXXXL 5.0114 0.0020 794 80 16 POL 503KIPMGVGL XIXXXXXL 795 80 16 POL 503 KIPMGVGLSPF XIXXXXXXXXF 796 85 17NUC 21 KLCLGWLW XLXXXXXW 797 85 17 NUC 21 KLCLGWLWGM XLXXXXXXXM 3.0209 *798 95 19 POL 489 KLHLYSHPI XLXXXXXXI 3.0009 * 799 80 16 POL 489KLHLYSHPII XLXXXXXXXI 800 80 16 POL 489 KLHLYSHPIIL XLXXXXXXXXL 801 7515 POL 108 KLIMPARF XLXXXXXF 802 75 15 POL 108 KLIMPARFY XLXXXXXXY1.0171 803 80 16 POL 610 KLPVNRPI XLXXXXXI 804 80 16 POL 610 KLPVNRPIDWXLXXXXXXXW 805 95 19 POL 574 KTKRWGYSL XTXXXXXXL 5.0034 806 85 17 POL574 KTKRWGYSLNF XTXXXXXXXXF 807 85 17 POL 620 KVCQRIVGL XVXXXXXXL 1.0198808 85 17 POL 620 KVCQRIVGLL XVXXXXXXXL 1.0567 809 95 19 POL 55 KVGNFTGLXVXXXXXL 17.0116 810 95 19 POL 55 KVGNFTGLY XVXXXXXXY 1.0166 * 811 85 17X 91 KVLHKRTL XVXXXXXL 812 85 17 X 91 KVLHKRTLGL XVXXXXXXXL 1.0800 813100 20 POL 121 KYLPLDKGI XYXXXXXXI 5.0063 * 0.0028 814 85 17 POL 745KYTSFPWL XYXXXXXL 17.0132 815 85 17 POL 745 KYTSFPWLL XYXXXXXXL 2.0061 *3.6000 816 80 16 ENV 247 LFILLLCL XFXXXXXL 17.0247 817 80 16 ENV 247LFILLLCLI XFXXXXXXI 818 80 16 ENV 247 LFILLLCLIF XFXXXXXXXF 819 80 16ENV 247 LFILLLCLIFL XFXXXXXXXXL 820 100 20 ENV 254 LIFLLVLL XIXXXXXLChisari 821 4.014 95 19 ENV 254 LIFLLVLLDY XIXXXXXXXY 1.0899 822 100 20POL 109 LIMPARFY XIXXXXXY 26.0028 823 95 19 POL 514 LLAQFTSAI XLXXXXXXI3.0010 * 824 100 20 ENV 251 LLCLIFLL XLXXXXXL Chisari 825 4.015 100 20ENV 251 LLCLIFLLVL XLXXXXXXXL 1.0898 826 100 20 ENV 251 LLCLIFLLVLLXLXXXXXXXXL Chisari 827 4.016 85 17 NUC 30 LLDTASAL XLXXXXXL 828 85 17NUC 30 LLDTASALY XLXXXXXXY 1.0155 * 829 95 19 ENV 260 LLDYQGML XLXXXXXLChisari 830 4.021 80 16 POL 752 LLGCAANW XLXXXXXW 831 80 16 POL 752LLGCAANWI XLXXXXXXI 3.0013 832 80 16 POL 752 LLGCAANWIL XLXXXXXXXL1.0912 * 833 95 19 POL 628 LLGFAAPF XLXXXXXF 834 75 15 ENV 63LLGWSPQAQGI XLXXXXXXXXI 835 100 20 ENV 250 LLLCLIFL XLXXXXXL Chisari 8364.017 100 20 ENV 250 LLLCLIFLL XLXXXXXXL 1.0834 * 837 100 20 ENV 250LLLCLIFLLVL XLXXXXXXXXL Chisari 838 4.018 100 20 ENV 378 LLPIFFCLXLXXXXXL 17.0112 839 100 20 ENV 378 LLPIFFCLW XLXXXXXXW 840 100 20 ENV378 LLPIFFCLWVY XLXXXXXXXXY 26.0549 * 841 95 19 NUC 44 LLSFLPSDFXLXXXXXXF 842 95 19 NUC 44 LLSFLPSDFF XLXXXXXXXF 843 95 19 POL 563LLSLGIHL XLXXXXXL 844 90 18 POL 407 LLSSNLSW XLXXXXXW 845 90 18 POL 407LLSSNLSWL XLXXXXXXL 1.0184 * 846 90 18 POL 407 LLSSNLSWLSL XLXXXXXXXXL847 80 16 ENV 184 LLTRILTI XLXXXXXI Chisari 848 4.053 80 16 POL 436LLVGSSGL XLXXXXXL 849 95 19 ENV 257 LLVLLDYQGM XLXXXXXXXM 3.0207 850 9519 ENV 257 LLVLLDYQGML XLXXXXXXXXL 851 95 19 ENV 175 LLVLQAGF XLXXXXXF852 95 19 ENV 175 LLVLQAGFF XLXXXXXXF 20.0121 853 90 18 ENV 175LLVLQAGFFL XLXXXXXXXL 1.0892 * 854 90 18 ENV 175 LLVLQAGFFLL XLXXXXXXXXLChisari 855 4.028 100 20 ENV 338 LLVPFVQW XLXXXXXW 856 100 20 ENV 338LLVPFVQWF XLXXXXXXF 857 90 18 NUC 100 LLWFHISCL XLXXXXXXL 1.0844 * 85885 17 NUC 100 LLWFHISCLTF XLXXXXXXXXF 859 95 19 POL 643 LMPLYACIXMXXXXXI 17.0130 860 75 15 NUC 137 LTFGRETVL XTXXXXXXL 861 75 15 NUC 137LTFGRETVLEY XTXXXXXXXXY 862 90 18 ENV 189 LTIPQSLDSW XTXXXXXXXW 863 9018 ENV 189 LTIPQSLDSWW XTXXXXXXXXW 864 90 18 POL 404 LTNLLSSNL XTXXXXXXL865 90 18 POL 404 LTNLLSSNLSW XTXXXXXXXXW 866 80 16 ENV 185 LTRILTIPQSLXTXXXXXXXXL 867 85 17 POL 99 LTVNEKRRL XTXXXXXXL 868 95 19 ENV 258LVLLDYQGM XVXXXXXXM 3.0034 869 95 19 ENV 258 LVLLDYQGML XVXXXXXXXL1.0515 870 95 19 ENV 176 LVLQAGFF XVXXXXXF 871 90 16 ENV 176 LVLQAGFFLXVXXXXXXL 1.0827 872 90 18 ENV 176 LVLQAGFFLL XVXXXXXXXL 1.0893 * 873100 20 ENV 339 LVPPVQWF XVXXXXXF 874 95 19 ENV 339 LVPFVQWFVGLXVXXXXXXXXL 875 90 18 NUC 119 LVSFGVWI XVXXXXXI Chisari 876 4.078 100 20POL 377 LVVDFSQF XVXXXXXF 877 90 18 NUC 101 LWFHISCL XWXXXXXL 878 85 17NUC 101 LWFHISCLTF XWXXXXXXXF 26.0373 879 85 17 NUC 27 LWGMDIDPYXWXXXXXXY 880 100 20 POL 151 LWKAGILY XWXXXXXY 881 80 16 POL 492LYSHPIIL XYXXXXXL 882 80 16 POL 492 LYSHPIILGF XYXXXXXXXF 2.0161 *1.1000 883 85 17 ENV 360 MMWYWGPSL XMXXXXXXL 1.0839 * 0.0012 884 85 17ENV 360 MMWYWGPSLY XMXXXXXXXY 1039.01 * 0.0001 885 85 17 ENV 361MWYWGPSL XWXXXXXL 17.0249 886 85 17 ENV 361 MWYWGPSLY XWXXXXXXY 1039.020.0027 887 95 19 POL 561 NFLLSLGI XFXXXXXI 888 95 19 POL 561 NFLLSLGIHLXFXXXXXXXL 5.0115 0.0099 889 95 19 POL 42 NLGNLNVSI XLXXXXXXI 3.0008 89095 19 POL 42 NLGNLNVSIPW XLXXXXXXXXW 891 90 18 POL 406 NLLSSNLSWXLXXXXXXW 892 90 18 POL 406 NLLSSNLSWL XLXXXXXXXL 1.0549 893 95 19 POL45 NLNVSIPW XLXXXXXW 894 100 20 POL 400 NLQSLTNL XLXXXXXL 895 100 20 POL400 NLQSLTNLL XLXXXXXXL 1.0189 896 75 15 ENV 15 NLSVPNPL XLXXXXXL 897 7515 ENV 15 NLSVPNPLGF XLXXXXXXXF 898 80 16 POL 758 NWILRGTSF XWXXXXXXF899 80 16 POL 758 NWILRGTSFVY XWXXXXXXXXY 900 95 19 POL 512 PFLLAQFTSAIXFXXXXXXXXI 901 95 19 POL 634 PFTQCGYPAL XFXXXXXXXL 5.0116 0.0002 902 9519 POL 634 PFTQCGYPALM XFXXXXXXXXM 903 95 19 ENV 341 PFVQWFVGL XFXXXXXXL5.0059 0.0003 904 85 17 POL 616 PIDWKVCQRI XIXXXXXXXI Chisari 905 4.091100 20 ENV 380 PIFFCLWVY XIXXXXXXY 1.0843 906 100 20 ENV 380 PIFFCLWVYIXIXXXXXXXI 20.0258 907 85 17 POL 713 PIHTAELL XIXXXXXL 908 80 16 POL 496PIILGFRKI XIXXXXXXI 927.48 909 80 15 POL 496 PIILGFRKlPM XIXXXXXXXXM 910100 20 ENV 314 PIPSSWAF XIXXXXXF 911 100 20 POL 124 PLDKGIKPY XLXXXXXXY1.0174 * 912 100 20 POL 124 PLDKGIKPYY XLXXXXXXXY 1.0541 * 913 95 19 POL20 PLEEELPRL XLXXXXXXL 1.0163 914 95 19 ENV 10 PLGFFPDHQL XLXXXXXXXL1.0511 915 100 20 POL 427 PLHPAAMPHL XLXXXXXXXL 1.0550 916 100 20 POL427 PLHPAAMPHLL XLXXXXXXXXL 917 100 20 ENV 377 PLLPIFFCL XLXXXXXXL1.0842 * 918 100 20 ENV 377 PLLPIFFCLW XLXXXXXXXW 919 95 19 ENV 174PLLVLQAGF XLXXXXXXF 920 95 19 ENV 174 PLLVLQAGFF XLXXXXXXXF 921 90 18ENV 174 PLLVLQAGFFL XLXXXXXXXXL Chisari 922 4.029 80 16 POL 711PLPIHTAEL XLXXXXXXL 1.0201 923 80 18 POL 711 PLPIHTAELL XLXXXXXXXL1.0569 924 75 16 POL 2 PLSYQHFRKL XLXXXXXXXL 1.0527 925 75 15 POL 2PLSYQHFRKLL XLXXXXXXXXL 926 85 17 POL 98 PLTVNEKRRL XLXXXXXXXL 1.0536927 80 16 POL 505 PMGVGLSPF XMXXXXXXF 928 80 16 POL 505 PMGVGLSPFLXMXXXXXXXL 1.0557 929 80 16 POL 505 PMGVGLSPFLL XMXXXXXXXXL 930 75 15POL 692 PTGWGLAI XTXXXXXI 931 85 17 POL 797 PTTGRTSL XTXXXXXL 932 85 17POL 797 PTTGRTSLY XTXXXXXXY 1.0208 * 933 80 16 NUC 15 PTVQASKL XTXXXXXL934 80 16 NUC 15 PTVQASKLCL XTXXXXXXXL 935 75 15 ENV 361 PTVWLSVIXTXXXXXI 936 75 15 ENV 351 PTVWLSVIW XTXXXXXXW 937 75 15 ENV 351PTVWLSVIWM XTXXXXXXXM 938 85 17 POL 612 PVNRPIDW XVXXXXXW 939 80 16 POL750 PWLLGCAANW XWXXXXXXXW 940 80 15 POL 750 PWLLGCAANWI XWXXXXXXXXI 941100 20 POL 51 PWTHKVGNF XWXXXXXXF 20.0138 * 0.0280 942 80 16 X 8QLDPARDVL XLXXXXXXL 1.0210 943 80 16 X 8 QLDPARDVLCL XLXXXXXXXXL Chisari944 4.073 90 18 NUC 99 QLLWFHISCL XLXXXXXXXL 1.0908 * 945 95 19 POL 665QVFADATPTGW XVXXXXXXXXW 946 95 19 ENV 344 QWFVGLSPTVW XWXXXXXXXX 947 7515 ENV 242 RFIIFLFI XFXXXXXI 17.0151 948 75 15 ENV 242 RFIIFLFILXFXXXXXXL 949 75 15 ENV 242 RFIIFLFILL XFXXXXXXXL 950 75 15 ENV 242RFIIFLFILLL XFXXXXXXXXL 951 100 20 ENV 332 RFSWLSLL XFXXXXXL 952 100 20ENV 332 RFSWLSLLVPF XFXXXXXXXXF 953 80 16 ENV 167 RILTIPQSL XIXXXXXXL1.0149 954 90 18 POL 524 RIVGLLGF XIXXXXXF 955 75 15 POL 106 RLKLIMPARFXLXXXXXXXF 956 75 15 POL 106 RLKLIMPARFY XLXXXXXXXXY 957 95 19 POL 376RLVVDFSQF XLXXXXXXF 20.0122 958 90 18 POL 355 RTPARVTGGVF XTXXXXXXXXF959 95 19 POL 36 RVAEDLNL XVXXXXXL 960 90 18 POL 36 RVAEDLNLGNLXVXXXXXXXXL 961 80 16 POL 818 RVHFASPL XVXXXXXL 962 100 20 POL 357RVTGGVFL XVXXXXXL 963 85 17 POL 577 RWGYSLNF XWXXXXXF 964 85 17 POL 577RWGYSLNFM XWXXXXXXM 965 85 17 POL 577 RWGYSLNRMGY XWXXXXXXXXY 966 95 19ENV 238 RWMCLRRF XWXXXXXF 967 95 19 ENV 236 RWMCLRRFI XWXXXXXXI20.0135 * 0.0710 968 95 19 ENV 236 RWMCLRRFII XWXXXXXXXI 20.0269 *1.1000 969 95 19 ENV 236 RWMCLRRRIF XWXXXXXXXXF 970 100 20 POL 167SFCGSPYSW XFXXXXXXW 20.0139 * 0.0710 971 95 19 NUC 46 SFLPSDFF XFXXXXXF972 80 16 POL 765 SFVYVPSAL XFXXXXXXL 973 100 20 POL 49 SIPWTHKVGNFXIXXXXXXXXF 974 95 19 ENV 194 SLDSWWTSL XLXXXXXXL 1.0150 975 95 19 ENV194 SLDSWWTSLNF XLXXXXXXXXF 976 95 19 POL 416 SLDVSAAF XLXXXXXF 977 9519 POL 416 SLDVSAAFY XLXXXXXXY 1.0186 * 978 100 20 ENV 337 SLLVPFVQWXLXXXXXXW 979 100 20 ENV 337 SLLVPFVQWF XLXXXXXXXF 980 75 15 POL 581SLNFMGYVI XLXXXXXXI 3.0011 981 95 19 X 54 SLRGLPVCAF XLXXXXXXXF 20.0259982 90 18 POL 403 SLTNLLSSNL XLXXXKXXXL 1.0548 983 75 15 X 104 STTDLEAYXTXXXXXY 984 75 15 X 104 STTDLEAYF XTXXXXXXF 985 75 15 ENV 17 SVPNPLGFXVXXXXXF 986 85 17 POL 548 SVQHLESL XVXXXXXL 987 80 16 ENV 330 SVRFSWLSLXVXXXXXXL 1.0153 988 80 16 ENV 330 SVRFSWLSLL XVXXXXXXXL 1.0517 989 9018 POL 739 SVVLSRKY XVXXXXXY 26.0029 990 85 17 POL 739 SVVLSRKYTSFXVXXXXXXXXF 991 95 19 POL 524 SVVRRAFPHCL XVXXXXXXXXL 992 95 19 POL 413SWLSLDVSAAF XWXXXXXXXXF 993 100 20 ENV 334 SWLSLLVPF XWXXXXXXF 20.0136 *0.3900 994 95 19 POL 392 SWPKFAVPNL XWXXXXXXXL 20.0271 * 5.6000 995 10020 ENV 197 SWWTSLNF XWXXXXXF 996 95 19 ENV 197 SWWTSLNFL XWXXXXXXL20.0137 * 0.3800 997 90 18 POL 537 SYMDDVVL XYXXXXXL 998 75 15 POL 4SYQHFRKL XYXXXXXL 999 75 15 POL 4 SYQHFRKLL XYXXXXXXL 2.0042 0.0051 100075 15 POL 4 SVQHFRKLLL XYXXXXXXXL 2.0173 * 0.0660 1001 75 15 POL 4SYQHFRKLLLL XYXXXXXXXXL 1002 75 15 NUC 138 TFGRETVL XFXXXXXL 1003 75 15NUC 138 TFGRETVLEY XFXXXXXXXY 1004 75 15 NUC 138 TFGRETVLEYL XFXXXXXXXXL1005 95 19 POL 657 TFSPTYKAF XFXXXXXXF 5.0064 0.0060 1006 95 19 POL 657TFSPTYKAFL XFXXXXXXKL 5.0117 0.0043 1007 90 18 ENV 190 TIPQSLDSWXIXXXXXXW 1008 90 18 ENV 190 TIPQSLDSWW XIXXXXXXXW 1009 100 20 POL 150TLWKAGIL XLXXXXXL 1010 100 20 POL 150 TLWKAGILY XLXXXXXXY 1.0177 * 101175 15 X 105 TTDLEAYF XTXXXXXF 1012 85 17 POL 798 TTGRTSLY XTXXXXXY26.0030 1013 85 17 POL 100 TVNEKRRL XVXXXXXL 1014 80 16 NUC 16 TVQASKLCLXVXXXXXXL 1.0365 1015 80 16 NUC 16 TVQASKLCLGW XVXXXXXXXXW 1016 75 15ENV 352 TVWLSVIW XVXXXXXW 1017 75 15 ENV 352 TVWLSVIWM XVXXXXXXM 3.00351018 95 19 POL 686 VFADATPTGW XFXXXXXXXW 20.0272 * 0.0180 1019 75 15 X131 VFVLGGCRHKL XFXXXXXXXXL 1020 85 17 POL 543 VLGAKSVQHL XLXXXXXXXL1.0560 1021 90 18 X 133 VLGGCRHKL XLXXXXXXL 1.0220 1022 85 17 X 92VLHKRTLGL XLXXXXXXL 1.0391 1023 95 19 ENV 259 VLLDYQGM XLXXXXXM 17.01071024 95 19 ENV 259 VLLDYQGML XLXXXXXXL 1.0151 * 1025 95 19 ENV 177VLQAGFFL XLXXXXXL Chisari 1026 4.027 95 19 ENV 177 VLQAGFFLL XLXXXXXXL1.0828 * 1027 85 17 POL 741 VLSRKYTSF XLXXXXXXF 1028 85 17 POL 741VLSRKYTSFPW XLXXXXXXXXW 1029 80 16 POL 542 VVLGAKSVQHL XVXXXXXXXXL 103085 17 POL 740 VVLSRKYTSF XVXXXXXXXXF 20.0261 1031 95 19 POL 525VVRRAFPHCL XVXXXXXXXL 1.0558 1032 95 19 NUC 124 VWIRTPPAY XWXXXXXXY 103375 15 ENV 353 VWLSVIWM XWXXXXXM 1034 90 18 NUC 102 WFHISCLTF XFXXXXXXF13.0073 * 0.0300 1035 95 19 ENV 345 WFVGLSPTVW XFXXXXXXXW 20.0270 *0.0120 1036 95 19 ENV 345 WFVGLSPTVWL XFXXXXXXXXL 1037 80 16 POL 759WILRGTSF XIXXXXXF 1038 80 16 POL 759 WLRGTSFVY XIXXXXXXXY 1.0572 1039 9519 NUC 125 WIRTPPAY XIXXXXXY 26.0031 1040 80 16 POL 751 WLLGCAANWXLXXXXXXW 1041 80 16 POL 751 WLLGCAANWI XLXXXXXXXI Chisari 1042 4.104 8016 POL 751 WLLGCAANWIL XLXXXXXXXXL 1043 95 19 POL 414 WLSLDVSAAFXLXXXXXXXF 1044 95 19 POL 414 WLSLDVSAAFY XLXXXXXXXXY 26.0551 1045 10020 ENV 335 WLSLLVPF XLXXXXXF 1046 100 20 ENV 335 WLSLLVPFVQW XLXXXXXXXXW1047 85 17 NUC 26 WLWGMDIDPY XLXXXXXXXY 1.0774 * 1048 95 19 ENV 237WMCLRRFI XMXXXXXI 1049 95 19 ENV 237 WMCLRRFII XMXXXXXXI 3.0031 * 0.02301050 95 19 ENV 237 WMCLRRFIIF XMXXXXXXXF 20.0266 0.0013 1051 95 19 ENV237 WMCLRRFIIFL XMXXXXXXXXL Chisari 1052 4.024 85 17 ENV 359 WMMWYWGPSLXMXXXXXXXL 1.0901 * 0.0005 1053 85 17 ENV 359 WMMWYWGPSL XMXXXXXXXXY26.0552 * 1054 100 20 POL 52 WTHKVGNF XTXXXXXF 1055 95 19 POL 52WTHKVGNFTGL XTXXXXXXXXL 1056 95 19 ENV 198 WWTSLNFL XWXXXXXL 1057 95 17ENV 362 WYWGPSLY XYXXXXXY 3.0362 0.0001 1058 100 20 POL 147 YLHTLWKAGIXLXXXXXXXI 7.0066 * 1059 100 20 POL 147 YLHTLWKAGIL XLXXXXXXXXL 1060 10020 POL 122 YLPLDKGI XLXXXXXI 1061 100 20 POL 122 YLPLDKGIKPY XLXXXXXXXXY26.0553 1062 90 18 NUC 118 YLVSFGVW XLXXXXXW 1063 90 18 NUC 118YLVSFGVWI XLXXXXXI 3.0007 * 1064 85 17 POL 746 YTSFPWLL XTXXXXXL 1065411

TABLE X HBV B07 SUPER MOTIF (With binding information) C- ConservancyFrequency Protein Position Sequence P2 term Peptide AA Filed B*0702B*3501 B*5101 B*5301 B*5401 SEQ ID NO: 75 15 X 146 APCNFFTSA P A 9 106695 19 POL 633 APFTQCGY P Y 19.0013 8 0.0001 0.0012 0.0019 0.0002 0.00021067 95 19 POL 633 APFTQCGYPA P A 16.0180 10 * 0.0029 0.0001 0.00021.4000 1068 95 19 POL 633 APFTQCGYPAL P L 26.0554 11 * 0.2300 0.00100.0004 −0.0003 0.0093 1069 100 20 ENV 232 CPGYRWMCL P L 1308.21 9 107080 16 NUC 14 CPTVQASKL P L 9 1071 80 16 NUC 14 CPTVQASKLCL P L 11 107280 16 X 10 DPARDVLCL P L 9 1073 80 16 ENV 122 DPRVRGLY P L 8 1074 90 18POL 778 DPSRGRLGL P L 1147.01 9 * 0.0120 0.0001 0.0001 0.0001 0.00011075 90 18 NUC 33 DPYKEFGA P A 19.0008 8 0.0001 0.0001 0.0019 0.00020.0019 1076 75 15 ENV 130 FPAGGSSSGTV P V 11 1077 90 18 ENV 14 FPDHQLDPAP A 1308.23 9 * 1078 85 17 ENV 14 FPDHQLDPAF P F 20.0274 10 0.00020.0016 0.0003 0.0011 0.0021 1079 95 19 POL 530 FPHCLAFSY P Y 1145.08 9 *0.0001 0.5250 0.0665 0.5400 0.0199 1080 95 19 POL 530 FPHCLAFSYM P M1147.05 10 * 0.0990 0.2200 0.0900 0.0790 0.0480 1081 75 15 POL 749FPWLLGCA P A 8 1082 75 15 POL 749 FPWLLGCAA P A 9 1083 75 15 POL 749FPWLLGCAANW P W 11 1084 90 18 X 67 GPCALRFTSA P A 16.0182 10 * 0.09000.0001 0.0001 0.0002 0.0035 1085 95 19 POL 19 GPLEEELPRL P L 15.0208 100.0001 0.0001 0.0002 0.0001 0.0002 1086 90 18 POL 19 GPLEEELPRLA P A26.0555 11 −0.0002 0.0001 0.0001 −0.0003 0.0001 1087 95 19 ENV 173GPLLVLQA P A 19.0003 8 * 0.0003 0.0001 0.0110 0.0002 0.0065 1088 95 19ENV 173 GPLLVLQAGF P A 15.0212 10 0.0001 0.0001 0.0002 0.0001 0.00021089 95 19 ENV 173 GPLLVLQAGFF P F 26.0556 11 0.0011 0.0001 0.00010.0008 0.0009 1090 85 17 POL 97 GPLTVNEKRRL P L 26.0557 11 0.0031 0.00010.0001 −0.0003 0.0001 1091 100 20 POL 429 HPAAMPHL P L 19.0011 8 *0.0650 0.0004 0.3100 0.0037 0.0160 1092 100 20 POL 429 HPAAMPHLL P L1147.02 9 * 0.0980 0.0270 0.0110 0.0500 0.0120 1093 85 17 POL 429HPAAMPHLLV P V 20.0273 10 * 0.0160 0.0020 0.0078 0.0140 0.0170 1094 8016 POL 495 HPILGFRKI P I 10 1095 100 20 ENV 313 IPIPSSWA P A 19.0005 8 *0.0004 0.0004 0.0019 0.0002 0.0600 1096 100 20 ENV 313 IPIPSSWAF P F1145.04 9 * 0.1300 2.7679 2.3500 0.7450 0.0034 1097 80 16 ENV 313IPIPSSWAFA P A 16.0177 10 * 0.0013 0.0024 0.0014 0.4500 1098 80 16 POL504 IPMGVGLSPF P F 10 1099 80 16 POL 504 IPMGVGLSPFL P L 11 1100 90 18ENV 191 IPQSLDSW P W F126.65 8 1101 90 18 ENV 191 IPQSLDSWW P W F126.609 * 1102 80 16 ENV 315 IPSSWAFA P A 8 1103 100 20 POL 50 IPWTHKVGNF P F15.0209 10 0.0013 0.0001 0.0007 0.0001 0.0002 1104 100 20 ENV 379LPIFFCLW P W 19.0007 8 * 0.0001 0.0001 0.0360 0.1400 0.0035 1105 100 20ENV 379 LPIFFCLWV P V 1308.22 9 * 1106 100 20 ENV 379 LPIFFCLWVY P Y15.0215 10 0.0002 0.0079 0.0002 0.0006 0.0002 1107 100 20 ENV 379LPIFFCLWVYI P I 26.0558 11 0.0002 0.0001 0.0043 0.0139 0.0021 1108 85 17POL 712 LPIHTAEL P L 17.0259 8 1109 85 17 POL 712 LPIHTAELL P L 20.01409 * 0.0040 0.0630 0.0052 0.3100 0.0005 1110 85 17 POL 712 LPIHTAELLA P A16.0181 10 * 0.0018 0.0011 0.0016 0.3300 1111 85 17 POL 712 LPIHTAELLAAP A 26.0559 11 0.0090 0.0027 −0.0003 0.0120 2.7500 1112 80 16 X 89LPKVLHKRTL P L 10 1113 100 20 POL 123 LPLDKGIKPY P Y 15.0210 10 * 0.00010.0290 0.0002 0.0003 0.0002 1114 100 20 POL 123 LPLDKGIKPYY P Y 26.056011 −0.0002 0.0009 0.0001 0.0007 0.0001 1115 95 19 X 58 LPVCAFSSA P A1147.06 9 * 0.0480 0.0710 0.0110 0.0009 19.0000 1116 80 16 POL 611LPVNRPIDW P W 9 1117 80 16 POL 611 LPVNRPIDWKV P V 11 80 16 POL 433MPHLLVGSSGL P L 11 100 20 POL 1 MPLSYQHF P F 19.0010 8 * 0.0001 0.00970.0120 0.0370 0.0190 75 15 POL 1 MPLSYQHFRKL P L 11 90 18 POL 774NPADDPSRGRL P L 26.0561 11 * 0.0120 0.0001 0.0001 −0.0003 0.0001 95 19ENV 9 NPLGFFPDHQL P L 26.0562 11 0.0012 0.0021 0.0001 0.0028 0.0001 7515 POL 571 NPNKTKRW P W 8 75 15 POL 571 NPNKTKRWGY P Y 10 95 19 NUC 129PPAYRPPNA P A 16.0007 9 0.0001 0.0001 0.0001 0.0002 0.0003 95 19 NUC 129PPAYRPPNAPI P I 26.0583 11 0.0003 0.0001 0.0001 −0.0003 0.0001 85 17 ENV58 PPHGGLLGW P W 20.0141 9 0.0001 0.0002 0.0001 0.0003 0.0002 100 20 NUC134 PPNAPILSTL P L 15.0211 10 0.0001 0.0001 0.0035 0.0001 0.0002 80 16POL 615 RPIDWKVCQRI P I 11 100 20 NUC 133 RPPNAPIL P L 19.0009 8 *0.0076 0.0001 0.0280 0.0002 0.0002 100 20 NUC 133 RPPNAPILSTL P L26.0564 11 * 0.1300 0.0001 0.0018 −0.0003 0.0001 100 20 NUC 44SPEHCSPHHTA P A 26.0565 11 −0.0002 0.0001 0.0001 −0.0003 0.0011 95 19POL 511 SPFLLAQF P F 19.0012 8 * 0.5500 0.0009 0.0180 0.0009 0.0093 9519 POL 511 SPFLLAQFTSA P A 26.0566 11 * 0.0820 0.0001 0.0001 −0.000312.0500 100 20 NUC 49 SPHHTALRQA P A 16.0178 10 0.0012 0.0001 0.00020.0035 100 20 NUC 49 SPHHTALRQAI P I 26.0567 11 * 0.5800 0.0001 0.00040.0005 0.0002 85 17 ENV 67 SPQAQGIL P L 8 85 17 POL 808 SPSVPSHL P L 875 15 ENV 350 SPTVWLSV P V 8 75 15 ENV 350 SPTVWLSVI P I 1308.16 9 75 15ENV 350 SPTVWLSVIW P W 1308.17 10 75 15 ENV 350 SPTVWLSVIWM P M 11 95 19POL 659 SPTYKAFL P L 19.0015 8 * 0.3900 0.0001 0.0019 0.0002 0.0002 9018 POL 354 TPARVTGGV P V 1147.07 9 * 0.0078 0.0001 0.0013 0.0001 0.001590 18 POL 354 TPARVTGGVF P F 1147.04 10 * 0.3200 0.1000 0.0001 0.00990.0006 90 18 POL 354 TPARVTGGVFL P L 26.0568 11 * 0.0950 0.0001 0.00010.0005 0.0005 95 19 NUC 128 TPPAYRPPNA P A 16.0179 10 * 0.0001 0.00010.0002 0.0100 75 15 ENV 57 TPPHGGLL P L 8 75 15 ENV 57 TPPHGGLLGW P W1308.04 10 80 16 POL 691 TPTGWGLA P A 8 75 15 POL 691 TPTGWGLAI P I 9 9519 ENV 340 VPFVQWFV P V 19.0008 8 * 0.0010 0.0001 19.0000 0.0002 0.110095 19 ENV 340 VPFVQWFVGL P L 15.0213 10 0.0011 0.0001 0.0100 0.00010.0025 95 19 POL 398 VPNLQSLTNL P L 15.0216 10 0.0006 0.0001 0.00040.0001 0.0002 95 19 POL 398 VPNLQSLTNLL P L 26.0569 11 0.0004 0.00010.0001 −0.0003 0.0002 90 18 POL 769 VPSALNPA P A 19.0016 8 * 0.00110.0001 0.0070 0.0002 1.0000 95 19 POL 393 WPKFAVPNL P L 15.0035 9 0.00540.0002 0.0016 0.0001 0.0015 95 19 POL 640 YPALMPLY P Y 19.0014 8 *0.0004 0.2600 0.4100 0.0450 0.0056 95 19 POL 640 YPALMPLYA P A 1147.089 * 0.0180 0.0480 0.0340 0.0140 16.0000 95 19 POL 640 YPALMPLYACI P I26.0570 11 0.0040 0.0001 0.0470 0.0320 0.0700 96

TABLE XI HBV B27 SUPER MOTIF Super- SEQ ID Source Conservancy FreqProtein Position Sequence String Motif Peptide Filed NO: HBV 95 19 X 51AHLSLRGL XHXXXXXL B27s 1162 HBV 85 17 POL 546 AKSVQHLESL XKXXXXXXXL B27s1163 HBV 90 18 POL 356 ARVTGGVF XRXXXXXF B27s 1164 HBV 90 18 POL 356ARVTGGVFL XRXXXXXXL B27s 1165 HBV 95 19 X 48 DHGAHLSL XHXXXXXL B27s 1166HBV 95 19 X 48 DHGAHLSLRGL XHXXXXXXXXL B27s 1167 HBV 90 18 ENV 16DHQLDPAF XHXXXXXF B27s 1168 HBV 100 20 POL 126 DKGIKPYY XKXXXXXY B27s1169 HBV 100 20 NUC 46 EHCSPHHTAL XHXXXXXXXL B27s 1170 HBV 90 18 NUC 103FHISCLTF XHXXXXXF B27s 1171 HBV 80 16 POL 501 FRKIPMGVGL XRXXXXXXXL B27s1172 HBV 80 16 POL 608 FRKLPVNRPI XRXXXXXXXI B27s 1173 HBV 75 15 NUC 140GRETVLEY XRXXXXXY B27s 1174 HBV 75 15 NUC 140 GRETVLEYL XRXXXXXXL B27s1175 HBV 100 20 NUC 51 HHTALRQAI XHXXXXXXI B27s 1176 HBV 95 19 NUC 51HHTALRQAIL XHXXXXXXXL B27s 1177 HBV 95 19 POL 54 HKVGNFTGL XKXXXXXXLB27s 17.0358 1178 HBV 95 19 POL 54 HKVGNFTGLY XKXXXXXXXY B27s 1179 HBV75 15 POL 568 IHLNPNKTKRW XHXXXXXXXXW B27s 1180 HBV 85 17 POL 714IHTAELLAACF XHXXXXXXXXF B27s 1181 HBV 85 17 POL 576 KRWGYSLNF XRXXXXXFB27s 1182 HBV 85 17 POL 576 KRWGYSLNFM XRXXXXXXXM B27s 1183 HBV 90 18 X93 LHKRTLGL XHXXXXXL B27s 1184 HBV 95 19 POL 490 LHLYSHPI XHXXXXXI B27s1185 HBV 80 16 POL 490 LHLYSHPII XHXXXXXXI B27s 1186 HBV 80 16 POL 490LHLYSHPIIL XHXXXXXXXL B27s 1187 HBV 100 20 POL 428 LHPAAMPHL XHXXXXXXLB27s 1188 HBV 100 20 POL 428 LHPAAMPHLL XHXXXXXXXL B27s 1189 HBV 100 20POL 148 LHTLWKAGI XHXXXXXXI B27s 1190 HBV 100 20 POL 148 LHTLWKAGILXHXXXXXXXL B27s 1191 HBV 100 20 POL 148 LHTLWKAGILY XHXXXXXXXXY B27s1192 HBV 75 15 POL 107 LKLIMPARF XKXXXXXXF B27s 1193 HBV 75 15 POL 107LKLIMPARFY XKXXXXXXXY B27s 1194 HBV 95 19 X 55 LRGLPVCAF XRXXXXXXF B27s1195 HBV 80 16 POL 761 LRGTSFVY XRXXXXXY B27s 1196 HBV 95 19 NUC 55LRQAILCW XRXXXXXW B27s 1197 HBV 90 18 NUC 55 LRQAILCWGEL XRXXXXXXXXLB27s 1198 HBV 95 19 ENV 240 LRRFIIFL XRXXXXXL B27s 1199 HBV 75 15 ENV240 LRRFIIFLF XRXXXXXXF B27s 1200 HBV 75 15 ENV 240 LRRFIIFLFIXRXXXXXXXI B27s 1201 HBV 75 15 ENV 240 LRRFIIFLFIL XRXXXXXXXXL B27s 1202HBV 75 15 POL 573 NKTKRWGY XKXXXXXY B27s 1203 HBV 75 15 POL 573NKTKRWGYSL XKXXXXXXXL B27s 1204 HBV 85 17 POL 34 NRRVAEDL XRXXXXXL B27s1205 HBV 85 17 POL 34 NRRVAEDLNL XRXXXXXXXL B27s 1206 HBV 95 19 POL 531PHCLAFSY XHXXXXXY B27s 1207 HBV 95 19 POL 531 PHCLAFSYM XHXXXXXXM B27s1208 HBV 85 17 ENV 59 PHGGLLGW XHXXXXXW B27s 1209 HBV 100 20 NUC 50PHHTALRQAI XHXXXXXXXI B27s 1210 HBV 95 19 NUC 50 PHHTALRQAIL XHXXXXXXXXLB27s 1211 HBV 80 16 POL 434 PHLLVGSSGL XHXXXXXXXL B27s 1212 HBV 95 19POL 394 PKFAVPNL XKXXXXXL B27s 1213 HBV 95 19 POL 394 PKFAVPNLQSLXKXXXXXXXXL B27s 1214 HBV 85 17 X 90 PKVLHKRTL XKXXXXXXL B27s 1215 HBV85 17 X 90 PKVLHKRTLGL XKXXXXXXXXL B27s 1216 HBV 75 15 POL 6 QHFRKLLLXHXXXXXL B27s 1217 HBV 75 15 POL 6 QHFRKLLLL XHXXXXXXL B27s 1218 HBV 9018 POL 623 QRIVGLLGF XRXXXXXXF B27s 1219 HBV 100 20 POL 145 RHYLHTLWXHXXXXXW B27s 1220 HBV 80 16 POL 502 RKIPMGVGL XKXXXXXXL B27s 1221 HBV80 16 POL 609 RKLPVNRPI XKXXXXXXI B27s 1222 HBV 80 16 POL 609RKLPVNRPIDW XKXXXXXXXXW B27s 1223 HBV 85 17 POL 744 RKYTSFPW XKXXXXXWB27s 1224 HBV 85 17 POL 744 RKYTSFPWL XKXXXXXXL B27s 1225 HBV 85 17 POL744 RKYTSFPWLL XKXXXXXXXL B27s 1226 HBV 95 19 POL 527 RRAFPHCL XRXXXXXLB27s 1227 HBV 95 19 POL 527 RRAFPHCLAF XRXXXXXXXF B27s 1228 HBV 75 15ENV 241 RRFIIFLF XRXXXXXF B27s 1229 HBV 75 15 ENV 241 RRFIIFLFIXRXXXXXXI B27s 1230 HBV 75 15 ENV 241 RRFIIFLFIL XRXXXXXXXL B27s 1231HBV 75 15 ENV 241 RRFIIFLFILL XRXXXXXXXXL B27s 1232 HBV 75 15 POL 105RRLKLIMPARF XRXXXXXXXXF B27s 1233 HBV 90 16 POL 35 RRVAEDLNL XRXXXXXXLB27s 1234 HBV 80 16 POL 494 SHPIILGF XHXXXXXF B27s 1235 HBV 80 16 POL494 SHPIILGFRKI XHXXXXXXXXI B27s 1236 HBV 90 18 NUC 20 SKLCLGWL XKKXXXXLB27s 1237 HBV 85 17 NUC 20 SKLCLGWLW XKXXXXXXW B27s 1238 HBV 85 17 NUC20 SKLCLGWLWGM XKXXXXXXXXM B27s 1239 HBV 85 17 POL 743 SRKYTSFPWXRXXXXXXW B27s 1240 HBV 85 17 POL 743 SRKYTSFPWL XRXXXXXXXL B27s 1241HBV 85 17 POL 743 SRKYTSFPWLL XRXXXXXXXXL B27s 1242 HBV 95 19 POL 375SRLVVDFSQF XRXXXXXXXF B27s 1243 HBV 80 16 POL 472 SRNLYVSL XRXXXXXL B27s17.0123 1244 HBV 95 19 POL 53 THKVGNFTGL XHXXXXXXXL B27s 1245 HBV 95 19POL 53 THKVGNFTGLY XHXXXXXXXXY B27s 1246 HBV 95 19 POL 575 TKRWGYSLXKXXXXXL B27s 1247 HBV 85 17 POL 575 TKRWGYSLNF XKXXXXXXXF B27s 1248 HBV85 17 POL 575 TKRWGYSLNFM XKXXXXXXXXM B27s 1249 HBV 100 20 POL 120TKYLPLDKGI XKXXXXXXXI B27s 1250 HBV 100 20 POL 144 TRHYLHTL XRXXXXXLB27s 1251 HBV 100 20 POL 144 TRHYLHTLW XRXXXXXXW B27s 1252 HBV 80 16 ENV186 TRILTIPQSL XRXXXXXXXL B27s 1253 HBV 80 16 POL 819 VHFASPLHVAWXHXXXXXXXXW B27s 1254 HBV 80 16 ENV 331 VRFSWLSL XRXXXXXL B27s 1255 HBV80 16 ENV 331 VRFSWLSLL XRXXXXXXL B27s 1256 HBV 95 19 POL 526 VRRAFPHCLXRXKXXXXL B27s 1257 HBV 95 19 POL 526 VRRAFPHCLAF XRXXXXXXXXF B27s 1258HBV 85 17 POL 619 WKVCQRIVGL XKXXXXXXXL B27s 1259 HBV 85 17 POL 619WKVCQRIVGLL XKXXXXXXXXL B27s 1260 HBV 100 20 NUC 132 YRPPNAPI XRXXXXXIB27s 1261 HBV 100 20 NUC 132 YRPPNAPIL XRXXXXXXL B27s 17.0356 1262 HBV95 19 ENV 235 YRWMCLRRF XRXXXXXXF B27s 1263 HBV 95 19 ENV 235 YRWMCLRRFIXRXXXXXXXI B27s 1264 HBV 95 19 ENV 235 YRWMCLRRFII XRXXXXXXXXI B27s 1265104

TABLE XII HBV B44 SUPER MOTIF Source Conservancy Freq Protein PositionSequence String Supermotif Peptide Filed SEQ ID NO: HBV 95 19 POL 688ADATPTGW XDXXXXXW B44 1266 HBV 95 19 POL 688 ADATPTGWGL XDXXXXXXXL B441267 HBV 80 16 POL 688 ADATPTGWGL XDXXXXXXXXA B44 1268 HBV 90 18 POL 776ADDPSRGRL XDXXXXXXL B44 1269 HBV 90 18 POL 776 ADDPSRGRLGL XDXXXXXXXXLB44 1270 HBV 95 19 POL 38 AEDLNLGNL XEXXXXXXL B44 17.0357 1271 HBV 95 19POL 38 AEDLNLGNLNV XEXXXXXXXXV B44 1272 HBV 85 17 POL 717 AELLAACFXEXXXXXF B44 1273 HBV 85 17 POL 717 AELLAACFA XEXXXXXXA B44 1274 HBV 9018 POL 777 DDPSAGRL XDXXXXXL B44 17.0010 1275 HBV 90 18 POL 777DDPSRGRLGL XDXXXXXXXL B44 17.0418 1276 HBV 90 18 POL 540 DDVVLGAKSVXDXXXXXXXV B44 1277 HBV 75 15 POL 18 DEAGPLEEEL XEXXXXXXXL B44 1278 HBV95 19 POL 39 EDUNLGNL XDXXXXXL B44 1279 HBV 95 19 POL 39 EDUNLGNUNVXDXXXXXXXV B44 1280 HBV 90 18 POL 22 EEELPRLA XEXXXXXA B44 1281 HBV 8016 X 121 EELGEEIRL XEXXXXXXL B44 1282 HBV 90 18 NUC 32 IDPYKEFGAXDXXXXXXA B44 1283 HBV 85 17 POL 617 IDWKVCORI XDXXXXXXI B44 1284 HBV 8517 POL 617 IDWKVCORIV XDXXXXXXXV B44 1285 HBV 100 20 POL 125 LDKGIKPYXDXXXXXY B44 1286 HBV 100 20 POL 125 LDKGIKPYY XDXXXXXXY B44 1287 HBV 8016 X 9 LDPARDVL XDXXXXXL B44 17.0012 1288 HBV 80 16 X 9 LDPARDVLCLXDXXXXXXXL B44 17.0419 1289 HBV 95 19 ENV 195 LDSWWTSL XDXXXXXL B44 1290HBV 95 19 ENV 195 LDSWWTSLNF XDXXXXXXXF B44 1291 HBV 90 18 BW 195LDSWWTSLNFL XDXXXXXXXXL B44 1292 HBV 85 17 NUC 31 LDTASALY XDXXXXXY B441293 HBV 80 16 NUC 31 LDTASALYREA XDXXXXXXXXA B44 1294 HBV 95 19 POL 417LDVSAAFY XDXXXXXY B44 1295 HBV 90 18 ENV 261 LDYQGMLPV XDXXXXXXV B441296 HBV 95 19 POL 21 LEEELPRL XEXXXXXL B44 1297 HBV 90 18 POL 21LEEELPRLA XEXXXXXXA B44 1298 HBV 90 18 POL 539 MDDVVLGA XDXXXXXA B441299 HBV 90 18 POL 539 MDDVVGAKSV XDXXXXXXXXV B44 1300 HBV 90 18 NUC 30MDIDPYKEF XDXXXXXXF B44 1301 HBV 90 18 NUC 30 MDIDPYKEFGA XDXXXXXXXXAB44 1302 HBV 95 19 ENV 15 PDHQLDPA XDXXXXXA B44 1303 HBV 90 18 ENV 15PDHQLDPAF XDXXXXXXF B44 1304 HBV 100 20 NUC 45 PEHCSPHHTA XEXXXXXXXA B441305 HBV 100 20 NUC 45 PEHCSPHHTAL XEXXXXXXXXL B44 1306 HBV 85 17 NUC 28RDLLDTASA XDXXXXXXA B44 1307 HBV 85 17 NUC 28 RDLLDTASAL XDXXXXXXXL B441308 HBV 85 17 NUC 28 RDLLDTASALY XDXXXXXXXXY B44 1309 HBV 95 19 X 13RDVLCLRPV XDXXXXXXV B44 1310 HBV 95 19 X 13 RDVLCLRPVGA XDXXXXXXXXA B441311 HBV 75 15 NUC 141 RETVLEYL XEXXXXXL B44 1312 HBV 75 15 NUC 141RETVLEYLV XEXXXXXXV B44 1313 HBV 90 18 POL 736 TDNSVVLSRKY XDXXXXXXXXYB44 1314 HBV 95 19 NUC 42 VELLSFLPSDF XEXXXXXXXXF B44 1315 HBV 80 16 X120 WEELGEEEI XEXXXXXI B44 1316 HBV 80 16 X 120 WEELGEEIRL XEXXXXXXXLB44 1317 52

TABLE XIII HBV B58 SUPER MOTIF Source Convervancy Freq Protein PositionSequence String Supermotif Peptide Filed SEQ ID NO: HBV 85 17 POL 431AAMPHLLV XAXXXXXV B58 1318 HBV 95 19 POL 632 AAPFTQCGY XAXXXXXXY B581319 HBV 85 17 NUC 34 ASALYREAL XSXXXXXXL B58 1320 HBV 100 20 POL 166ASFCGSPY XSXXXXXY B58 26.0026 * 1321 HBV 100 20 POL 166 ASFCGSPYSWXSXXXXXXXW B58 1322 HBV 90 18 NUC 19 ASKLCLGW XSXXXXXW B58 1323 HBV 9018 NUC 19 ASKLCLGWL XSXXXXXXL B58 1324 HBV 85 17 NUC 19 ASKLCLGWLWXSXXXXXXXW B58 1325 HBV 80 16 POL 822 ASPLHVAW XSXXXXXW B58 1326 HBV 8016 ENV 329 ASVRFSWL XSXXXXXL B58 1327 HBV 80 16 ENV 329 ASVRFSWLSLXSXXXXXXXL B58 1328 HBV 80 16 ENV 329 ASVRFSWLSLL XSXXXXXXXXL B58 1329HBV 95 19 POL 690 ATPTGWGL XTXXXXXL B58 1330 HBV 75 15 POL 690ATPTGWGLAI XTXXXXXXXI B58 1331 HBV 95 19 X 61 CAFSSAGPCAL XAXXXXXXXXLB58 1332 HBV 100 20 NUC 48 CSPHHTAL XSXXXXXL B58 1333 HBV 80 16 POL 471CSRNLYVSL XSXXXXXXL B58 1334 HBV 95 19 POL 523 CSVVRRAF XSXXXXXF B581335 HBV 100 20 ENV 310 CTCIPIPSSW XTXXXXXXXW B58 1336 HBV 95 19 POL 689DATPTGWGL XAXXXXXXL B58 5.0027 1337 HBV 75 15 POL 689 DATPTGWGLAIXAXXXXXXXXI B58 1338 HBV 95 19 ENV 196 DSWWTSLNF XSXXXXXXF B58 20.01201339 HBV 90 18 ENV 196 DSWWTSLNFL XSXXXXXXXL B58 1340 HBV 80 16 NUC 32DTASALYREA XTXXXXXXXXL B58 1341 HBV 80 16 NUC 32 DTASALYREAL XTXXXXXXXXLB58 1342 HBV 100 20 POL 17 EAGPLEEEL XAXXXXXXL B58 5.0028 1343 HBV 95 19POL 374 ESRLVVDF XSXXXXXF B58 1344 HBV 95 19 POL 374 ESRLVVDFSQFXSXXXXXXXXF B58 1345 HBV 75 15 NUC 142 ETVLEYLV XTXXXXXV B58 1346 HBV 9519 POL 631 FAAPFTQCGY XAXXXXXXXV B58 20.0254 * 1347 HBV 95 19 POL 687FADATPTGW XAXXXXXXW B58 1348 HBV 95 19 POL 687 FADATPTGWGL XAXXXXXXXXLB58 1349 HBV 80 16 POL 821 FASPLHVAW XAXXXXXXW B58 1350 HBV 95 19 POL396 FAVPNLQSL XAXXXXXXL B58 5.0029 * 1351 HBV 95 19 POL 658 FSPTYKAFXSXXXXXF B58 1352 HBV 95 19 POL 658 FSPTYKAFL XSXXXXXXL B58 1353 HBV 9519 X 63 FSSAGPCAL XSXXXXXXL B58 1354 HBV 90 18 X 63 FSSAGPCALRFXSXXXXXXXXF B58 1355 HBV 100 20 ENV 333 FSWLSLLV XSXXXXXV B58 1356 HBV100 20 ENV 333 FSWLSLLVPF XSXXXXXXXF B58 20.0263 1357 HBV 100 20 ENV 333FSWLSLLVPFV XSXXXXXXXXV B58 1358 HBV 90 18 POL 536 FSYMDDVV XSXXXXXV B5817.0257 1359 HBV 90 18 POL 536 FSYMDDVVL XSXXXXXXL B58 1360 HBV 95 19POL 656 FTFSPTYKAF XTXXXXXXXF B58 20.0262 1361 HBV 95 19 POL 656FTFSPTYKAFL XTXXXXXXXXL B58 1362 HBV 90 18 POL 59 FTGLYSSTV XTXXXXXXVB58 20.0118 1363 HBV 95 19 POL 635 FTQCGYPAL XTXXXXXXL B58 5.0031 1364HBV 95 19 POL 635 FTQCGYPALM XTXXXXXXXM B58 5.0085 1365 HBV 95 19 POL518 FTSAICSV XTXXXXXV B58 1366 HBV 95 19 POL 518 FTSAICSVV XTXXXXXXV B585.0032 1367 HBV 95 19 X 50 GAHLSLRGL XAXXXXXXL B58 5.0040 1368 HBV 90 18X 50 GAHLSLRGLPV XAXXXXXXXXV B58 1369 HBV 85 17 POL 545 GAKSVQHLXAXXXXXL B58 1370 HBV 85 17 POL 545 GAKSVQHLESL XAXXXXXXXXL B58 1371 HBV75 15 ENV 134 GSSSGTVNPV XSXXXXXXXV B58 1372 HBV 90 18 POL 735 GTDNSVVLXTXXXXXL B58 1373 HBV 75 15 ENV 13 GTNLSVPNPL XTXXXXXXXL B58 1374 HBV 8016 POL 763 GTSFVYVPSAL XTXXXXXXXXL B58 1375 HBV 85 17 POL 715 HTAELLAACFXTXXXXXXXF B58 1376 HBV 100 20 NUC 52 HTALRQAI XTXXXXXI B58 1377 HBV 9519 NUC 52 HTALRQAIL XTXXXXXXL B58 5.0021 1378 HBV 95 19 NUC 52HTALRQAILCW XTXXXXXXXXW B58 1379 HBV 100 20 POL 149 HTLWKAGI XTXXXXXIB58 1380 HBV 100 20 POL 149 HTLWKAGIL XTXXXXXXL B58 5.0033 1381 HBV 10020 POL 149 HTLWKAGILY XTXXXXXXXY B58 1.0542 * 1382 HBV 90 18 NUC 105ISCLTFGRETV XSXXXXXXXXV B58 1383 HBV 85 17 POL 547 KSVQHLESL XSXXXXXXLB58 1384 HBV 95 19 POL 574 KTKRWGYSL XTXXXXXXL B58 5.0034 1385 HBV 85 17POL 574 KTKRWGYSLNF XTXXXXXXXXF B58 1386 HBV 90 18 POL 534 LAFSYMDDVXAXXXXXXV B58 20.0118 1387 HBV 90 18 POL 534 LAFSYMDDVV XAXXXXXXXV B5820.0257 1388 HBV 90 18 POL 534 LAFSYMDDVVL XAXXXXXXXXL B58 1389 HBV 9519 POL 515 LAQFTSAI XAXXXXXI B58 1390 HBV 95 19 POL 515 LAQFTSAICSVXAXXXXXXXXV B58 1391 HBV 95 19 NUC 45 LSFLPSDF XSXXXXXF B58 1392 HBV 9519 NUC 45 LSFLPSDFF XSXXXXXXF B58 20.0123 1393 HBV 95 19 POL 415LSLDVSAAF XSXXXXXXF B58 1394 HBV 95 19 POL 415 LSLDVSAAFY XSXXXXXXXY B582.0239 * 1395 HBV 100 20 ENV 336 LSLLVPFV XSXXXXXV B58 1396 HBV 100 20ENV 336 LSLLVPFVQW XSXXXXXXXW B58 1397 HBV 100 20 ENV 336 LSLLVPFVQWFXSXXXXXXXXF B58 1398 HBV 95 19 X 53 LSLRGLPV XSXXXXXV B58 1399 HBV 95 19X 53 LSLRGLPVCAF XSXXXXXXXXF B58 1400 HBV 95 19 POL 510 LSPFLLAQFXSXXXXXXF B58 1401 HBV 75 15 ENV 349 LSPTVWLSV XSXXXXXXV B58 1402 HBV 7515 ENV 349 LSPTVWLSVI XSXXXXXXXI B58 1403 HBV 75 15 ENV 349 LSPTVWLSVIWXSXXXXXXXXW B58 1404 HBV 85 17 POL 742 LSRKYTSF XSXXXXXF B58 1405 HBV 8517 POL 742 LSRKYTSFPW XSXXXXXXXW B58 1406 HBV 85 17 POL 742 LSRKYTSFPWLXSXXXXXXXXL B58 1407 HBV 90 18 POL 408 LSSNLSWL XSXXXXXL B58 1408 HBV 9018 POL 408 LSSNLSWLSL XSXXXXXXXL B58 1409 HBV 100 20 NUC 140 LSTLPETTVXSXXXXXXV B58 1410 HBV 100 20 NUC 140 LSTLPETTVV XSXXXXXXXV B58 1411 HBV75 15 ENV 16 LSVPNPLGF XSXXXXXXF B58 1412 HBV 100 20 POL 412 LSWLSLDVXSXXXXXV B58 1413 HBV 75 15 POL 3 LSYQHFRKL XSXXXXXXL B58 1414 HBV 75 15POL 3 LSYQHFFRKLL XSXXXXXXXL B58 1415 HBV 75 15 POL 3 LSYQHFRKLLLXSXXXXXXXXL B58 1416 HBV 95 19 NUC 108 LTFGRETV XTXXXXXV B58 1417 HBV 7515 NUC 137 LTFGRETVL XTXXXXXXL B58 1418 HBV 75 15 NUC 137 LTFGRETVLEYXTXXXXXXXXY B58 1419 HBV 90 18 ENV 189 LTIPQSLDSW XTXXXXXXXW B58 1420HBV 90 18 ENV 189 LTIPQSLDSWW XTXXXXXXXXW B58 1421 HBV 90 18 POL 404LTNLLSSNL XTXXXXXXL B58 1422 HBV 90 18 POL 404 LTNLLSSNLSW XTXXXXXXXXWB58 1423 HBV 80 16 ENV 185 LTRILTIPQSL XTXXXXXXXXL B58 1424 HBV 85 17POL 99 LTVNEKRRL XTXXXXXXL B58 1425 HBV 75 15 X 103 MSTTDLEAY XSXXXXXXYB58 2.0126 * 1426 HBV 75 15 X 103 MSTTDLEAYF XSXXXXXXXF B58 1427 HBV 10020 NUC 136 NAPILSTL XAXXXXXL B58 1428 HBV 90 18 POL 738 NSVVLSRKYXSXXXXXXY B58 2.0123 1429 HBV 100 20 POL 430 PAAMPHLL XAXXXXXL B58 1430HBV 85 17 POL 430 PAAMPHLLV XAXXXXXXV B58 1431 HBV 90 18 POL 775PADDPSRGRL XAXXXXXXXL B58 1432 HBV 90 18 ENV 131 PAGGSSSGTV XAXXXXXXXVB58 1433 HBV 95 19 POL 641 PALMPLYACI XAXXXXXXXI B58 5.0087 1434 HBV 8016 X 11 PARDVLCL XAXXXXXL B58 1435 HBV 75 15 X 11 PARDVLCLRPVXAXXXXXXXXV B58 1436 HBV 90 18 POL 355 PARVTGGV XAXXXXXV B58 1437 HBV 9018 POL 355 PARVTGGVF XAXXXXXXF B58 1438 HBV 90 18 POL 355 PARVTGGVFLXAXXXXXXXL B58 1439 HBV 90 18 POL 355 PARVTGGVFLV XAXXXXXXXXV B58 1440HBV 95 19 NUC 130 PAYRPPNAPI XAXXXXXXXI B58 5.0081 1441 HBV 95 19 NUC130 PAYRPPNAPIL XAXXXXXXXXL B58 1442 HBV 90 18 POL 779 PSRGRLGL XSXXXXXLB58 1443 HBV 75 15 POL 692 PTGWGLAI XTXXXXXI B58 1444 HBV 85 17 POL 797PTTGRTSL XTXXXXXL B58 1445 HBV 85 17 POL 797 PTTGRTSLY XTXXXXXXY B581.0208 * 1446 HBV 80 16 NUC 15 PTVQASKL XTXXXXXL B58 1447 HBV 80 16 NUC15 PTVQASKLCL XTXXXXXXXL B58 1448 HBV 75 15 ENV 351 PTVWLSVI XTXXXXXIB58 1449 HBV 75 15 ENV 351 PTVWLSVIW XTXXXXXXW B58 1450 HBV 150 30 ENV351 PTVWLSVIWM XTXXXXXXXM B58 1451 HBV 95 19 POL 654 QAFTFSPTY XAXXXXXXYB58 20.0127 1452 HBV 80 16 ENV 179 QAGFFLLTRIL XAXXXXXXXXL B58 1453 HBV90 18 NUC 57 QAILCWGEL XAXXXXXXL B58 1454 HBV 180 36 NUC 57 QAILCWGELMXAXXXXXXXM B58 1455 HBV 80 16 ENV 107 QAMQWNSTTF XAXXXXXXXF B58 1456 HBV80 16 NUC 18 QASKLCLGW XAXXXXXXW B58 1457 HBV 80 16 NUC 18 QASKLCLGWLXAXXXXXXXL B58 1458 HBV 75 15 NUC 18 QASKLCLGWLW XAXXXXXXXXW B58 1459HBV 90 18 ENV 193 QSLDSWWTSL XSXXXXXXXL B58 F126.63 1460 HBV 90 18 POL402 QSLTNLLSSNL XSXXXXXXXXL B58 1461 HBV 95 19 POL 528 RAFPHCLAFXAXXXXXXF B58 20.0125 1462 HBV 95 19 POL 528 RAFPHCLAFSY XAXXXXXXXXY B5826.0550 * 1463 HBV 90 18 POL 353 RTPARVTGGV XTXXXXXXXV B58 1464 HBV 9018 POL 353 RTPARVTGGVF XTXXXXXXXXF B58 1465 HBV 90 18 X 65 SAGPCALRFXAXXXXXXF B58 26.0152 1466 HBV 95 19 POL 520 SAICSVVRRAF XAXXXXXXXXF B581467 HBV 90 18 NUC 35 SALYREAL XAXXXXXL B58 1468 HBV 100 20 POL 165SASFCGSPY XAXXXXXXY B58 20.0117 * 1469 HBV 100 20 POL 165 SASFCGSPYSWXAXXXXXXXXW B58 1470 HBV 95 19 X 64 SSAGPCAL XSXXXXXL B58 1471 HBV 90 18X 64 SSAGPCALRF XSXXXXXXXF B58 26.0374 1472 HBV 75 15 ENV 136 SSGTVNPVXSXXXXXV B58 1473 HBV 90 18 POL 409 SSNLSWLSL XSXXXXXXL B58 1474 HBV 9018 POL 409 SSNLSWSLDV XSXXXXXXXXV B58 1475 HBV 75 15 ENV 135 SSSGTVNPVXSXXXXXXV B58 1476 HBV 100 20 NUC 141 STLPETTV XTXXXXXV B58 1477 HBV 10020 NUC 141 STLPETTVV XTXXXXXXV B58 5.0024 1478 HBV 75 15 X 104 STTDLEAYXTXXXXXY B58 1479 HBV 75 15 X 104 STTDLEAYF XTXXXXXXF B58 1480 HBV 85 17POL 716 TAELLAACF XAXXXXXXF B58 1481 HBV 95 19 NUC 53 TALRQAIL XAXXXXXLB58 1482 HBV 95 19 NUC 53 TALRQAILCW XAXXXXXXXW B58 1483 HBV 80 16 NUC33 TASALYREAL XAXXXXXXXL B58 1484 HBV 95 19 POL 519 TSAICSVV XSXXXXXVB58 1485 HBV 80 16 POL 764 TSFVYVPSAL XSXXXXXXXL B58 1486 HBV 80 16 ENV168 TSGFLGPL XSXXXXXL B58 1487 HBV 75 15 ENV 168 TSGFLGPLL XSXXXXXXL B581488 HBV 75 15 ENV 168 TSGFLGPLLV XSXXXXXXXV B58 1489 HBV 75 15 ENV 168TSGFLGPLLVL XSXXXXXXXXL B58 1490 HBV 75 15 X 105 TTDLEAYF XTXXXXXF B581491 HBV 85 17 POL 798 TTGRTSLY XTXXXXXY B58 26.0030 1492 HBV 95 19 POL37 VAEDLNLGNL XAXXXXXXXL B58 5.0089 1493 HBV 100 20 POL 48 VSIPWTHKVXSXXXXXXV B58 1494 HBV 95 19 POL 391 VSWPKFAV XSXXXXXV B58 1495 HBV 9519 POL 391 VSWPKFAVPNL XSXXXXXXXXL B58 1496 HBV 100 20 POL 358 VTGGVFLVXTXXXXXV B58 1497 HBV 85 17 ENV 66 WSPQAQGI XSXXXXXI B58 1498 HBV 85 17ENV 66 WSPQAQGIL XSXXXXXXL B58 1499 HBV 100 20 POL 52 WTHKVGNF XTXXXXXFB58 1500 HBV 95 19 POL 52 WTHKVGNFTGL XTXXXXXXXXL B58 1501 HBV 80 16 POL493 YSHPIILGF XSXXXXXXF B58 1502 HBV 85 17 POL 580 YSLNFMGY XSXXXXXY B5826.0032 1503 HBV 75 15 POL 580 YSLNFMGYV XSXXXXXXV B58 1504 HBV 75 15POL 580 YSLNFMGYVI XSXXXXXXXI B58 1505 HBV 85 17 POL 746 YTSFPWLLXTXXXXXL B58 1506 189

TABLE XIV HBV B62 SUPER MOTIF Source Conservancy Freq Protein PositionSequence String Supermotif Peptide Filed SEQ ID NO: HBV 95 19 POL 521AICSVVRRAF XIXXXXXXXF B62s 1507 HBV 90 18 NUC 58 ALLCWGELM XIXXXXXXMB62s 1508 HBV 95 19 POL 642 ALMPLYACI XLXXXKXXI B62s 3.0012 * 1509 HBV95 19 NUC 54 ALRQAILCW XLXXXXXXW B62s 1510 HBV 80 16 ENV 108 AMQWNSTTFXMXXXXXXF B62s 1511 HBV 95 19 POL 633 APFTQCGY XPXXXXXY B62s 19.00131512 HBV 95 19 POL 516 AQFTSAICSV XQXXXXXXXV B62s 1513 HBV 95 19 POL 516AQFTSAICSVV XQXXXXXXXXV B62s 1514 HBV 100 20 ENV 312 CIPIPSSW XIXXXXXWB62s 1515 HBV 100 20 ENV 312 CIPIPSSWAF XIXXXXXXXF B62s 1516 HBV 90 16POL 533 CLAFSYMDDV XLXXXXXXXV B62s 1.0559 1517 HBV 90 18 POL 533CLAFSYMDDVV XLXXXXXXXXV B62s 1518 HBV 85 17 NUC 23 CLGWLWGM XLXXXXXMB62s 1519 HBV 85 17 NUC 23 CLGWLWGMDI XLXXXXXXXI B62s 2.0229 1520 HBV 9519 ENV 253 CLIFLLVLLDY XLXXXXXXXY B62s 26.0548 1521 HBV 95 19 ENV 239CLRRFIIF XLXXXXXF B62s 1522 HBV 75 15 ENV 239 CLRRFIIFLF XLXXXXXXXF B62s1523 HBV 75 15 ENV 239 CLRRFIIFLFI XLXXXXXXXXI B62s Chisari 1524 HBV 9015 NUC 107 CLTFGRETV XLXXXXXXV B62s 1.0160 1525 HBV 80 16 X 7 CQLDRADVXQXXXXXXV B62s 1526 HBV 85 17 POL 622 CQRIVGLLGF XQXXXXXXXF B62s 1527HBV 90 18 NUC 31 DIDPYKEF XIXXXXXF B62s 1528 HBV 85 17 NUC 29 DLLDTASALYXLXXXXXXXY B62s 1.0519 * 1529 HBV 95 19 POL 40 DLNLGNLNV XLXXXXXXV B62s1.0164 1530 HBV 95 19 POL 40 DLNLGNLNVSI XLXXXXXXXXI B62s 1531 HBV 80 16ENV 122 DPRVRGLY XPXXXXXY B62s 1532 HBV 95 19 X 14 DVLCLRPV XVXXXXXVB62s 1533 HBV 90 18 POL 541 DVVLGAKSV XVXXXXXXV B62s 1.0190 1534 HBV 9519 NUC 43 ELLSFLPSDF XLXXXXXXXF B62s 1535 HBV 95 19 NUC 43 ELLSFLPSDFFXLXXXXXXXXF B62s 1536 HBV 80 16 ENV 248 FILLLCLI XIXXXXXI B62s Chisari1537 HBV 80 16 ENV 248 FILLLCLIF XIXXXXXXF B62s 1538 HBV 80 16 ENV 246FIFLLLLCLI XLXXXXXXXI B62s 3.0206 1539 HBV 80 16 ENV 246 FLFILLLCLIFXLXXXXXXXXF B62s 1540 HBV 95 19 POL 513 FLLAQFTSAI XLXXXXXXXI B62s1147.13 * 1541 HBV 80 16 ENV 183 FLLTRILTI XLXXXXXXI B62s 3.0005 * 1542HBV 95 19 ENV 256 FLLVLLDY XLXXXXXY B62s 26.0027 1543 HBV 95 19 ENV 256FLLVLLDYQGM XLXXXXXXXXM B62s 1544 HBV 75 15 ENV 130 FPAGGSSSGTVXPXXXXXXXXV B62s 1545 HBV 85 17 ENV 14 FPDHQLDPAF XPXXXXXXXF B62s20.0274 1546 HBV 95 19 POL 530 FPHCLAFSY XPXXXXXXY B62s 15.0037 * 1547HBV 95 19 POL 530 FPHCLAFSYM XPXXXXXXXM B62s 15.0217 * 1548 HBV 75 15POL 749 FPWLLGCAANW XPXXXXXXXXW B62s 1549 HBV 95 19 ENV 346 FVGLSPTVXVXXXXXV B62s 1550 HBV 95 19 ENV 346 FVGLSPTVW XVXXXXXXW B62s 1551 HBV90 18 X 132 FVLGGCRHKLV XVXXXXXXXXV B62s 1552 HBV 95 19 POL 627GLLGFAAPF XLXXXXXXF B62s 20.0124 1553 HBV 95 19 POL 509 GLSPFLLAQFXLXXXXXXXF B62s 1554 HBV 75 15 ENV 348 GLSPTVWLSV XLXXXXXXXV B62s1.0518 * 1555 HBV 75 15 ENV 348 GLSPTVWLSVI XLXXXXXXXXI B62s Chisari1556 HBV 85 17 NUC 29 GMDIDPYKEF XMXXXXXXXF B62s 26.0372 1557 HBV 95 19ENV 173 GPLLVLQAGF XPXXXXXXXF B62s 15.0212 1558 HBV 95 19 ENV 173GPLLVLQAGFF XPXXXXXXXXF B62s 26.0556 1559 HBV 95 19 NUC 123 GVWIRTPPAYXVXXXXXXXY B62s 1.0525 1560 HBV 75 15 POL 569 HLNPNKTKRW XLXXXXXXXW B62s1561 HBV 90 18 X 52 HLSLRGLPV XLXXXXXXV B62s 1.0212 1562 HBV 80 16 POL491 HLYSHPII XLXXXXXI B62s 17.0256 1563 HBV 80 16 POL 491 HLYSHPIILGFXLXXXXXXXXF B62s 1564 HBV 85 17 POL 429 HPAAMPHLLV XPXXXXXXXV B62s20.0273 * 1565 HBV 80 16 POL 495 HPIILGFRKI XPXXXXXXXI B62s 1566 HBV 8016 POL 497 IILGFRKI XIXXXXXI B62s 17.0124 * 1567 HBV 80 16 POL 497IILGFRKIPM XIXXXXXXXM B62s 1568 HBV 90 18 NUC 59 ILCQGELM XLXXXXXM B62s1569 HBV 80 16 POL 498 ILGFRKIPM XLXXXXXXM B62s 3.0016 1570 HBV 100 20ENV 249 ILLLCLIF XLXXXXXF B62s 1571 HBV 100 20 ENV 249 ILLLCLIFLLVXLXXXXXXXXV B62s Chisari 1572 HBV 80 16 POL 760 ILRGTSFV XLXXXXXV B62s1573 HBV 80 16 POL 760 ILRGTSFVY XLXXXXXXY B62s 1.0205 * 1574 HBV 80 16POL 760 ILRGTSFVYV XLXXXXXXXV B62s 1.0573 * 1575 HBV 100 20 NUC 139ILSTLPETTV XLXXXXXXXV B62 1.0526 1576 HBV 100 20 NUC 139 ILSTLPETTVVXLXXXXXXXXV B62s * 1577 HBV 90 18 ENV 188 ILTIPQSLDSW XLXXXXXXXXW B62s1578 HBV 100 20 ENV 313 IPIPSSWAF XPXXXXXXF B62s 15.0032 * 1579 HBV 8016 POL 504 IPMGVGLSPF XPXXXXXXXF B62s 1580 HBV 90 18 ENV 191 IPQSLDSWXPXXXXXW B62s 19.0004 1581 HBV 90 18 ENV 191 IPQSLDSWW XPXXXXXXW B62s15.0030 * 1582 HBV 100 20 POL 50 IPWTHKVGNF XPXXXXXXXF B62s 15.0209 1583HBV 90 18 POL 625 IVGLLGFAAPF XVXXXXXKXXF B62s 1584 HBV 80 16 POL 503KIPMGVGLSPF XIXXXXXXXXF B62s 1585 HBV 85 17 NUC 21 KLCLGWLW XLXXXXXWB62s 1586 HBV 85 17 NUC 21 KLCLGWLWGM XLXXXXXXXM B62s 3.0209 * 1587 HBV95 19 POL 489 KLHLYSHPI XLXXXXXXI B62s 3.0009 * 1588 HBV 80 16 POL 489KLHLYSHPII XLXXXXXXXI B62s 1589 HBV 75 15 POL 108 KLIMPARF XLXXXXXF B62s1590 HBV 75 15 POL 108 KLIMPARFY XLXXXXXXY B62s 1.0171 1591 HBV 80 16POL 610 KLPVNRPI XLXXXXXI B62s 1592 HBV 80 16 POL 610 KLPVNRPIDWXLXXXXXXXW B62s 1593 HBV 95 19 POL 653 KQAFTFSPTY XQXXXXXXXY B62s20.0256 1594 HBV 95 19 POL 55 KVGNFTGLY XVXXXXXXY B62s 1.0166 * 1595 HBV95 19 ENV 254 LIFLLVLLDY XIXXXXXXXY B62s 1.0899 1596 HBV 100 20 POL 109LIMPARFY XIXXXXXY B62s 26.0028 1597 HBV 95 19 POL 514 LLAQFTSAIXLXXXXXXI B62s 3.0010 * 1598 HBV 100 20 ENV 251 LLCLIFLLV XLXXXXXXV B62s1.0835 * 1599 HBV 85 17 NUC 30 LLDTASALY XLXXXXXXY B62s 1.0155 * 1600HBV 90 18 ENV 260 LLDYQGMLPV XLXXXXXXXV B62s 1.0516 * 1601 HBV 80 16 POL752 LLGCAANW XLXXXXXW B62s 1602 HBV 80 16 POL 752 LLGCAANWI XLXXXXXXIB62s 3.0013 1603 HBV 95 19 POL 628 LLGFAAPF XLXXXXXF B62s 1604 HBV 75 15ENV 63 LLGWSPQAQGI XLXXXXXXXXI B62s 1605 HBV 100 20 ENV 250 LLLCLIFLLVXLXXXXXXXV B62s 1.0897 * 1606 HBV 100 20 ENV 378 LLPIFFCLW XLXXXXXXWB62s * 1607 HBV 100 20 ENV 378 LLPIFFCLWV XLXXXXXXXV B62s 1.0904 * 1608HBV 100 20 ENV 378 LLPIFFCLWVY XLXXXXXXXXY B62s 26.0549 1609 HBV 95 19NUC 44 LLSFLPSDF XLXXXXXXF B62s 1610 HBV 95 19 NUC 44 LLSFLPSDFFXLXXXXXXXF B62s 1611 HBV 90 18 POL 407 LLSSNLSW XLXXXXXW B62s 1612 HBV80 16 ENV 184 LLTRILTI XLXXXXXI B62s Chisari 1613 HBV 80 16 POL 436LLVGSSGL XLXXXXXL b62S 1614 HBV 95 19 ENV 257 LLVLLDYQGM XLXXXXXXXM B62s3.0207 1615 HBV 95 19 ENV 175 LLVLQAGF XLXXXXXF B62s 1616 HBV 95 19 ENV175 LLVLQAGFF XLXXXXXXF B62s 20.0121 1617 HBV 100 20 ENV 338 LLVPFVQWXLXXXXXW B62s 1618 HBV 100 20 ENV 338 LLVPFVQWF XLXXXXXXF B62s 1619 HBV95 19 ENV 338 LLVPFVQWFV XLXXXXXXXV B62s 1.0930 * 1620 HBV 85 17 NUC 100LLWFHISCLTF XLXXXXXXXXF B62s 1621 HBV 95 19 POL 643 LMPLYACI XMXXXXXIB62s 17.0130 1622 HBV 100 20 ENV 379 LPIFFCLW XPXXXXXW B62s 19.0007 1623HBV 100 20 ENV 379 LPIFFCLWV XPXXXXXXV B62s 15.0034 1624 HBV 100 20 ENV379 LPIFFCLWVY XPXXXXXXXY B62s 15.0215 1625 HBV 100 20 ENV 379LPIFFCLWVYI XPXXXXXXXXI B62s 26.0558 1626 HBV 100 20 POL 123 LPLDKGIKPYXPXXXXXXXY B62s 15.0210 * 1627 HBV 100 20 POL 123 LPLDKGIKPYYXPXXXXXXXXY B62s 26.0560 1628 HBV 80 16 POL 611 LPVNRPIDW XPXXXXXXW B62s1629 HBV 80 16 POL 611 LPVNRPIDWKV XPXXXXXXXXV B62s 1630 HBV 80 16 ENV178 LQAGFFLLTRI XQXXXXXXXXI B62s 1631 HBV 95 19 ENV 258 LVLLDYQGMXVXXXXXXM B62s 3.0034 1632 HBV 95 19 ENV 176 LVLQAGFF XVXXXXXF B62s 1633HBV 100 20 ENV 339 LVPFVQWF XVXXXXXF B62s 1634 HBV 95 19 ENV 339LVPFVQWFV XVXXXXXXV B62s 1.0877 * 1635 HBV 90 1 8 NUC 119 LVSFGVWIXVXXXXXI B62s Chisari 1636 HBV 100 20 POL 377 LWDFSQF XVXXXXXF B62s 1637HBV 85 17 ENV 360 MMWYWGPSLY XMXXXXXXXY B62s 1039.01 * 1638 HBV 100 20POL 1 MPLSYQHF XPXXXXXF B62s 19.0010 * 1639 HBV 60 16 ENV 109 MQWNSTTFXQXXXXXF B62s 1640 HBV 95 19 POL 42 NLGNLNVSI XLXXXXXXI B62s 3.0008 1641HBV 95 19 POL 42 NLGNLNVSIPW XLXXXXXXXXW B62s 1642 HBV 90 18 POL 406NLLSSNLSW XLXXXXXXW B62s 1643 HBV 95 19 POL 45 NLNVSIPW XLXXXXXW B62s1644 HBV 75 15 ENV 15 NLSVPNPLGF XLXXXXXXXF B62s 1645 HBV 90 18 POL 411NLSWLSLDV XLXXXXXXV B62s 1.0185 * 1646 HBV 75 15 POL 571 NPNKTKRWXPXXXXXW B62s 1647 HBV 75 15 POL 571 NPNKTKRWGY XPXXXXXXXY B62s 1648 HBV100 20 POL 47 NVSIPWTHKV XVXXXXXXXV B62s 1.0532 1649 HBV 85 17 POL 616PIDWKVCQRI XIXXXXXXXI B62s Chisari 1650 HBV 85 17 POL 616 PIDWKVCQRIVXIXXXXXXXXV B62s 1651 HBV 100 20 ENV 380 PIFFCLWV XIXXXXXV B62s 1652 HBV100 20 ENV 380 PIFFCLWVY XIXXXXXXY B62s 1.0843 1653 HBV 100 20 ENV 380PIFFCLWVYI XIXXXXXXXI B62s 20.0258 1654 HBV 80 16 POL 496 PIILGFRKIXIXXXXXXI B62s 927.48 1655 HBV 80 16 POL 496 PIILGFRKIPM XIXXXXXXXXMB62s 1656 HBV 100 20 NUC 138 PILSTLPETTV XIXXXXXXXXV B62s Chisari 1657HBV 100 20 ENV 314 PIPSSWAF XIXXXXXF B62s 1658 HBV 100 20 POL 124PLDKGIKPY XLXXXXXXY B62s 1.0174 * 1659 HBV 100 20 POL 124 PLDKGIKPYYXLXXXXXXXY B62s 1.0541 * 1660 HBV 100 20 ENV 377 PLLPIFFCLW XLXXXXXXXWB62s 1661 HBV 100 20 ENV 377 PLLPIFFCLWV XLXXXXXXXXV B62s 1662 HBV 95 19ENV 174 PLLVLQAGF XLXXXXXXF B62s 1663 HBV 95 19 ENV 174 PLLVLQAGFFXLXXXXXXXF B62s 1664 HBV 80 16 POL 505 PMGVGLSPF XMXXXXXXF B62s 1665 HBV95 19 NUC 129 PPAYRPPNAPI XPXXXXXXXXI B62s 26.0563 1666 HBV 85 17 ENV 58PPHGGLLGW XPXXXXXXW B62s 20.0141 1667 HBV 80 16 ENV 106 PQAMQWNSTTXQXXXXXXXXF B62s 1668 HBV 90 18 ENV 192 PQSLDSWW XQXXXXXW B62s 1669 HBV85 17 POL 612 PVNRPIDW XVXXXXXW B62s 1670 HBV 85 17 POL 612 PVNRPIDWKVXVXXXXXXXV B62s 1.0566 1671 HBV 80 16 X 8 QLDPARDV XLXXXXXV B62s Chisari1672 HBV 95 19 POL 685 QVFADATPTGW XVXXXXXXXXW B62s 1673 HBV 90 18 POL624 RIVGLLGF XIXXXXXF B62s 1674 HBV 75 15 POL 106 RLKLIMPARF XLXXXXXXXFB62s 1675 HBV 75 15 POL 106 RLKLIMPARFY XLXXXXXXXXY B62s 1676 HBV 95 19POL 376 RLWDFSQF XLXXXXXXF B62s 20.0122 1677 HBV 80 16 POL 615RPIDWKVCORI XPXXXXXXXXI B62s 1678 HBV 90 18 NUC 56 RQAILCWGELMXQXXXXXXXXM B62s 1679 HBV 90 18 NUC 98 RQLLWFHI XQXXXXXI B62s 1680 HBV75 15 POL 818 RVHFASPLHV XVXXXXXXXV B62s 1.0576 1681 HBV 100 20 POL 357RVTGGVFLV XVXXXXXXV B62s 1.0181 1682 HBV 100 20 POL 49 SIPWTHKV XIXXXXXVB62s 1683 HBV 100 20 POL 49 SIPWTHKVGNF XIXXXXXXXXF B62s 1684 HBV 95 19ENV 194 SLDSWWTSLNF XLXXXXXXXXF B62s 1685 HBV 95 19 POL 416 SLDVSAAFXLXXXXXF B62s 1686 HBV 95 19 POL 416 SLDVSAAFY XLXXXXXXY B62s 1.0186 *1687 HBV 100 20 ENV 337 SLLVPFVQW XLXXXXXXW B62s 1688 HBV 100 20 ENV 337SLLVPFVQWF XLXXXXXXXF B62s 1689 HBV 95 19 ENV 337 SLLVPFVQWFVXLXXXXXXXXV B62s 1690 HBV 75 15 POL 581 SLNFMGYV XLXXXXXV B62s 1691 HBV75 15 POL 581 SLNFMGYVI XLXXXXXXI B62s 3.0011 1692 HBV 95 19 X 54SLRGLPVCAF XLXXXXXXXF B62s 20.0259 1693 HBV 95 19 POL 511 SPFILAQFXPXXXXXF B62s 19.0012 * 1694 HBV 100 20 NUC 49 SPHHTALRQAI XPXXXXXXXXIB62s 26.0567 * 1695 HBV 75 15 ENV 350 SPTVWLSV XPXXXXXV B62s 1696 HBV 7515 ENV 350 SPTVWLSVI XPXXXXXXI B62s 1308.16 1697 HBV 75 15 ENV 350SPTVWLSVIW XPXXXXXXXW B62s 1308.17 1698 HBV 75 15 ENV 350 SPTVWLSVIWMXPXXXXXXXXM B62s 1699 HBV 75 15 ENV 17 SVPNPLGF XVXXXXXF B62s 1700 HBV80 16 ENV 330 SVRFSWLSLLV XVXXXXXXXXV B62s 1701 HBV 90 18 POL 739SWLSRKY XVXXXXXY B62s 26.0029 1702 HBV 85 17 POL 739 SVVLSRKYTSFXVXXXXXXXXF B62s 1703 HBV 90 18 ENV 190 TIPQSLDSW XIXXXXXXW B62s 1704HBV 90 18 ENV 190 TIPQSLDSWW XIXXXXXXXW B62s 1705 HBV 100 20 NUC 142TLPETTVV XLXXXXXV B62s 1706 HBV 100 20 POL 150 TLWKAGILY XLXXXXXXY B62s1.0177 * 1707 HBV 90 18 POL 354 TPARVTGGV XPXXXXXXV B62s 15.0033 * 1708HBV 90 18 POL 35 TPARVTGGVF XPXXXXXXXF B62s 15.0214 * 1709 HBV 75 15 ENV57 TPPHGGLLGW XPXXXXXXXW B62s 1308.04 1710 HBV 75 15 POL 691 TPTGWGLAIXPXXXXXXI B62s 1711 HBV 95 19 POL 636 TQCGVPALM XQXXXXXXM B62s 1712 HBV80 16 NUC 16 TVQASKLCLGW XVXXXXXXXXW B62s 1713 HBV 75 15 ENV 352TVWLSVIW XVXXXXXW B62s 1714 HBV 75 15 ENV 352 TVWLSVIWM XVXXXXXXM B62s3.0035 1715 HBV 90 18 X 133 VLGGCRHKLV XLXXXXXXXV B62s 1.0589 1716 HBV95 19 ENV 259 VLLDYQGM XLXXXXXM B62s 17.0107 1717 HBV 90 18 ENV 259VLLDYQGMLPV XLXXXXXXXXV B62s 1147.14 * 1718 HBV 85 17 POL 741 VLSRKYTSFXLXXXXXXF B62s 1719 HBV 85 17 POL 741 VLSRKYTSFPW XLXXXXXXXXW B62s 1720HBV 95 19 ENV 340 VPFVQWFV XPXXXXXV B62s 19.0006 * 1721 HBV 80 16 NUC 17VQASKLCLGW XQXXXXXXXW B62s 1722 HBV 95 19 ENV 343 VQWFVGLSPTVXQXXXXXXXXV B62s 1723 HBV 90 18 POL 542 VVLGAKSV XVXXXXXV B62s 1724 HBV80 16 POL 759 WILRGTSF XIXXXXXF B62s 1725 HBV 80 16 POL 759 WILRGTSFVXIXXXXXXV B62s 1.0204 * 1726 HBV 80 16 POL 759 WILRGTSFVY XIXXXXXXXYB62s 1.0572 1727 HBV 80 16 POL 759 WILRGTSFVYV XIXXXXXXXXV B62s 1728 HBV95 19 NUC 125 WIRTPPAY XIXXXXXY B62s 26.0031 1729 HBV 80 16 POL 751WLLGCAANW XLXXXXXXW B62s 1730 HBV 80 16 POL 751 WLLGCAANWI XLXXXXXXXIB62s Chisari 1731 HBV 95 19 POL 414 WLSLDVSAAF XLXXXXXXXF B62s 1732 HBV95 19 POL 414 WLSLDVSAAFY XLXXXXXXXXY B62s 26.0551 1733 HBV 100 20 ENV335 WLSLLVPF XLXXXXXF B62s 1734 HBV 100 20 ENV 335 WLSLLVPFV XLXXXXXXVB62s 1.0838 * 1735 HBV 100 20 ENV 335 WLSLLVPFVQW XLXXXXXXXXW B62s 1736HBV 85 17 NUC 26 WLWGMDIDPY XLXXXXXXXY B62s 1.0774 * 1737 HBV 95 19 ENV237 WMCLRRFI XMXXXXXI B62s 1738 HBV 95 19 ENV 237 WMCLRRFII XMXXXXXXIB62s 3.0031 * 1739 HBV 95 19 ENV 237 WMCLRRFIIF XMXXXXXXXF B62s 20.02661740 HBV 85 17 ENV 359 WMMWyWGPSL XMXXXXXXXXY B62s 1.0901 * 1741 HBV 10020 POL 147 YLHTLWKAGI XLXXXXXXXI B62s 7.0066 1742 HBV 100 20 POL 122YLPLDKGI XLXXXXXI B62s 1743 HBV 100 20 POL 122 YLPLDKGIKPY XLXXXXXXXXYB62s 26.0553 1744 HBV 90 18 NUC 118 YLVSFGVW XLXXXXXW B62s 1745 HBV 9016 NUC 118 YLVSFGVWI XLXXXXXXI B62s 3.0007 * 1746 HBV 95 19 POL 640YPALMPLY XPXXXXXY B62s 19.0014 * 1747 HBV 95 19 POL 640 YPALMPLYACIXPXXXXXXXXI B62s 26.0570 1748 242

TABLE XV HBV A01 Motif (With Binding Information) 1st C- SEQ IDConservancy Frequency Protein Pos Sequence P2 term Peptide Filed A*0101NO: 80 16 ENV 119 AMQWNSTTF M F 1749 90 16 POL 748 DNSVVLSRKY N Y20.0255 0.0001 1750 95 19 POL 642 FAAPFTQCGY A Y 20.0254 * 0.0680 175185 17 POL 590 GYSLNFMGY Y Y 2.0058 1752 100 20 POL 149 HTLWKAGILY T Y1069.04 * 0.1100 1753 95 19 POL 664 KQAFTFSPTY Q Y 20.0256 0.0001 175485 17 NUC 30 LLDTASALY L Y 1069.01 * 12.0000 1755 95 19 POL 415LSLDVSAAFY S Y 1090.07 * 0.0150 1756 85 17 ENV 360 MMWYWGPSLY M Y1039.01 * 0.0810 1757 75 15 X 103 MSTTDLEAY S Y 2.0126 * 0.8500 1758 9018 POL 738 NSVVLSRKY S Y 2.0123 0.0005 1759 100 20 POL 124 PLDKGIKPY L Y1147.12 * 1760 100 20 POL 124 PLDKGIKPYY L Y 1069.03 * 0.1700 1761 85 17POL 797 PTTGRTSLY T Y 1090.09 * 0.2100 1762 100 20 POL 165 SASFCGSPY A Y1763 95 19 POL 416 SLDVSAAFY L Y 1069.02 * 5.2000 1764 16

TABLE XVI HBV A03 and A11 Motif (With binding information) ConservancyFrequency Protein 1st Pos Sequence Motif Super Motif P2 C-term PeptideFiled A*0301 A*1101 SEQ ID NO: 85 17 POL 721 AACFARSR A03/A11 A03 A R26.0003 0.0004 0.0003 1765 95 19 POL 643 AAPFTQCGY A03/A11 A03 A Y 176695 19 POL 540 AFPHCLAFSY A03/A11 A03 F Y 1767 95 19 X 62 AFSSAGPCAA03/A11 A03 F A 1768 95 19 POL 866 AFTFSPTYK A03/A11 A03 F K 20.0130 *0.2600 0.0400 1769 95 19 POL 666 AFTFSPTYKA A03/A11 A03 F A 1770 95 19POL 18 AGFLEEFLPR A03/A11 A03 G R 20.0265 0.0004 0.0002 1771 95 19 POL521 AICSVVRR A03/A11 A03 I R 26.0004 −0.0002 0.0003 1772 95 19 POL 532AICSVVRRAF A03/A11 A03 I F 1773 90 18 POL 772 ALNPADDPSR A03/A11 A03 L R1.1090 0.0003 0.0001 1774 85 17 X 70 ALRFTSAR A03/A11 A03 L R 26.00050.0047 0.0009 1775 80 16 ENV 119 AMQWNSTTF A03/A11 A03 M F 1776 80 16ENV 119 AMQWNSTTF A03/ A03 M F 1777 80 16 ENV 119 AMQWNSTTFH A03/A11 A03M H 1778 80 16 POL 822 ASPLHVAWR A03/A11 A03 S R 1779 75 15 ENV 84ASTNRCSGR A03/A11 A03 S R 1150.60 0.0009 0.0002 1780 80 16 POL 755CAANWILR A03/A11 A03 A R 1781 85 17 X 69 CALRFTSAR A03/A11 A03 A R26.0149 * 0.0034 0.0230 1782 85 17 POL 734 CFARSRSGA A03/A11 A03 F A1783 75 15 POL 618 CFRKLPVNR A03/A11 A03 F R 1784 95 19 POL 649CGYPALMPLY A03/A11 A03 G Y 1785 100 20 EVN 323 CIPIPSSWAF A03/A11 A03 IF 1786 90 18 X 17 CLRPVGAESR A03/A11 A03 L R 1.1093 0.0011 0.0001 178775 15 ENV 239 CLRRHFIIFLF A03/A11 A03 L F 1788 100 20 NUC 48 CSPHHTALRA03/A11 A03 S R 5.0055 * 0.0029 0.0001 1789 95 19 POL 534 CSVVRRAFPHA03/A11 A03 S H 1790 85 17 NUC 58 DLLDTASALY A03/A11 A03 L Y 1.0519 *0.0001 0.0001 1791 85 17 NUC 29 DLLDTASALYR A03/A11 A03 L R 26.05300.0042 −0.0003 1792 95 19 ENV 207 DSWWTSLNF A03/A11 A03 S F 20.01200.0006 0.0002 1793 85 17 NUC 32 DTASALYR A03/A11 A03 T R 26.0006 0.0004−0.0002 1794 95 19 POL 17 EAGPLEEELPR A03/A11 A03 A R 26.0531 −0.0009−0.0003 1795 90 18 POL 718 ELLAACFAR A03/A11 A03 L R 1.0988 0.00020.0004 1796 85 17 POL 718 ELLAACFARSR A03/A11 A03 L R 26.0532 0.00620.0016 1797 95 19 NUC 43 ELLSFLPSDF A03/A11 A03 L F 1798 95 19 NUC 72ESPEHCSPH A03/A11 A03 S H 1799 95 19 NUC 72 ESPEHCSPHH A03/A11 A03 S H1800 95 19 NUC 174 ETTVVRRR A03/A11 A03 T R 26.0007 0.0003 −0.0002 180180 16 NUC 174 ETTVVRRRGR A03/A11 A03 T R 1.1073 0.0003 0.0001 1802 95 19POL 642 FAAPFTQCGY A01/A03/ A03 A Y 20.0254 * 1803 80 16 POL 821FASPLHVAWR A03/A11 A03 A R 1804 90 18 ENV 24 FFPDHQLDPA A03/A11 A03 F A1805 75 15 NUC 139 FGRETVLEY A03/A11 A03 G Y 1806 75 15 POL 255FGVEPSGSGH A03/A11 A03 G H 1807 80 16 ENV 248 FILLLCLIF A03/A11 A03 I F1808 90 18 X 63 FSSAGPCALR A03/A11 A03 S R 1809 100 20 ENV 344FSWLSLLVPF A03/A11 A03 S F 20.0263 0.0004 0.0002 1810 95 19 POL 656FTFSPTYK A03/A11 A03 T K 1147.19 * 0.0100 0.0100 1811 95 19 POL 867FTFSPTYKAF A03/A11 A03 T F 20.0262 0.0004 0.0006 1812 95 19 POL 518FTSAICSVVR A03/A11 A03 T R 1.1085 0.0003 0.0003 1813 95 19 POL 518FTSAICSVVRR A03/A11 A03 T R 26.0533 0.0065 0.0092 1814 90 18 X 132FVLGGCRHK A03/A11 A03 V K 1090.03 * 0.0430 0.0090 1815 80 16 POL 765GCAANWILR A03/A11 A03 C R 1816 75 15 POL 587 GIHLNPNK A03/A11 A03 I K1817 75 15 POL 567 GIHLNPNKTK A03/A11 A03 I K 1.0563 0.0025 0.0011 181876 15 POL 567 GIHLNPNKTKR A03/A11 A03 I R 1819 95 19 POL 638 GLLGFAAPFA03/A11 A03 L F 20.0124 0.0006 0.0002 1820 95 19 POL 520 GLSPFLLAQFA03/A11 A03 L F 1821 85 17 NUC 29 GMDIDPYK A03/A11 A03 M K 26.00090.0006 0.0004 1822 85 17 NUC 29 GMDIDPYKEF A03/A24 A03 M F 26.0372−0.0003 −0.0002 1823 90 18 POL 735 GTDNSVVLSR A03/A11 A03 T R 1090.04 *0.0010 0.0420 1824 90 18 POL 735 GTDNSVVLSRK A03/A11 A03 T K 1147.17 *0.0140 0.5600 1825 80 16 POL 258 GVEPSGSGH A03/A11 A03 V H 1826 100 20POL 372 GVFLVDKNPH A03/A11 A03 V H 1827 95 19 NUC 152 GVWIRTPPAY A03/A11A03 V Y 1.0525 0.0047 0.0002 1828 95 19 NUC 123 GVVIRTPPAYR A03/A11 A03V R 26.0535 * 0.1900 0.1700 1829 100 20 NUC 78 HCSPHHTALR A03/A11 A03 CR 1830 80 18 POL 831 HFASPLHVA A03/A11 A03 F A 1831 90 18 NUC 104HISCLTFGR A03/A11 A03 I R 1069.18 * 0.0160 0.0065 1832 75 15 POL 569HLNPNKTK A03/A11 A03 L K 1833 75 15 POL 569 HLNPNKTKR A03/A11 A03 L H1.0983 0.0025 0.0001 1834 85 17 POL 728 HTAELLAACF A03/A11 A03 T F 1835100 20 POL 149 HTLWKAGILYK A03/A11 A03 T K 1147.16 * 0.5400 0.4400 183695 19 POL 533 ICSVVRRAF A03/A11 A03 C F 1837 95 19 ENV 266 IFLLVLLDYA03/A11 A03 F Y 1838 80 16 POL 771 ILRGTSFVY A03/A11 A03 L Y 1.0205 *0.0440 00002 1839 90 18 NUC 105 ISCLTFGR A03/A11 A03 S R 26.0010 0.00040.0002 1840 100 20 POL 153 KAGILYKR A03/A11 A03 A R 26.0011 0.0002−0.0002 1841 75 15 POL 108 KLIMPARFY A03/A11 A03 L Y 1.0171 1842 80 16POL 610 KLPVNRPIDWK A03/A11 A03 L K 1843 75 15 X 130 KVFVLGGCR A03/A11A03 V R 1.0993 * 0.0420 0.0820 1844 75 15 X 130 KVFVLGGCRH A03/A11 A03 VH 1845 95 19 POL 55 KVGNFTGLY A03/A11 A03 V Y 1142.05 * 0.2100 0.01701846 85 17 POL 720 LAACFARSR A03/A11 A03 A R 20.0129 0.0058 0.0065 1847100 20 POL 125 LDKGIKPYY A03/A11 A03 D Y 1848 95 19 ENV 206 LDSWWTSLNFA03/A11 A03 D F 1849 85 17 NUC 60 LDTASALYR A03/A11 A03 D R 26.01510.0004 −0.0002 1850 95 19 POL 428 LDVSAAFYH A03/A11 A03 D H 1851 80 16EVN 247 LFILLLCLIF A03/A11 A03 F F 1852 80 16 ENV 247 LFILLLCLIF A03/A24A03 F F 1853 80 16 POL 764 LGCAANWILR A03/A11 A03 G R 1854 75 15 POL 577LGHLNPNK A03/A11 A03 G K 1855 95 19 ENV 265 LIFLLVLLDY A03/A11 A03 I Y1.0899 0.0022 0.0004 1856 90 18 POL 719 LLAACFAR A03/A11 A03 L R 26.00120.0024 0.0003 1857 85 17 POL 719 LLAACFARSR A03/A11 A03 L R 1858 85 17NUC 30 LLDTASALYR A03/A11 A03 L R 1.1070 0.0050 0.0002 1859 80 16 POL752 LLGCAANWILR A03/A11 A03 L R 1860 95 19 NUC 44 LLSFLPSDF A03/A11 A03L F 1861 95 19 NUC 44 LLSFLPSDFF A03/A11 A03 L F 1862 95 19 ENV 175LLVLQAGFF A03/A11 A03 L F 20.0121 0.0006 0.0002 1863 100 20 ENV 349LLVPFVQWF A03/A11 A03 L F 1864 95 19 NUC 45 LSFLPSDFF A03/A11 A03 S F20.0123 0.0006 0.0002 1865 95 19 POL 426 LSLDVSAAF A03/A11 A03 S F 186675 15 POL 564 LSLGIHLNPNK A03/A11 A03 S K 1867 95 19 X 53 LSLRGLPVCAA03/A11 A03 S A 1868 95 19 POL 521 LSPFLLAQF A03/A11 A03 S F 1869 95 19NUC 169 LSTLPETTVVR A03/A11 A03 S R 26.0537 −0.0009 0.0008 1870 75 15ENV 18 LSVPNPLGF A03/A11 A03 S F 1871 100 20 POL 423 LSWLSLDVSA A03/A11A03 S A 20.0260 0.0048 0.0035 1872 75 15 POL 3 LSYQHFRK A03/A11 A03 S K1873 85 17 POL 99 LTVNEKRR A03/A11 A03 T R 26.0013 −0.0002 −0.0002 187490 18 NUC 119 LVSFGVWIR A03/A11 A03 V R 1090.08 * 0.0028 0.0120 1875 10020 POL 377 LVVDFSQFSR A03/A11 A03 V R 1069.20 * 0.0016 0.3600 1876 95 19ENV 249 MCLRRFIIF A03/A11 A03 C F 1877 90 18 POL 550 MDDVVLGAK A03/A11A03 D K 1878 90 18 NUC 30 MDIDIPYKEF A03/A11 A03 D F 1879 85 17 ENV 360MMWYWGPSLY A01/A03/ A03 M Y 1039.01 * 0.0500 0.0008 1880 75 15 X 103MSTTDLEAYF A03/A11 A03 S F 1881 75 15 X 103 MSTTDLEAYFK A03/A11 A03 S K1882 95 19 POL 572 NFLLSLGIH A03/A11 A03 F H 1883 90 18 NUC 75 NLEDPASRA03/A11 A03 L R 26.0014 −0.0002 −0.0002 1884 95 19 POL 45 NLNVSIPWTHA03/A11 A03 L H 1885 95 19 POL 45 NLNVSIPWTHK A03/A11 A03 L K 26.0538−0.0009 0.0005 1886 75 15 ENV 15 NLSVPNPLGF A03/A11 A03 L F 1887 75 15ENV 215 NSQSPTSNH A03/A11 A03 S H 1888 90 18 POL 738 NSVVLSRK A03/A11A03 S K 26.0015 0.0006 0.0010 1889 100 20 POL 47 NVSIPWTHK A03/A11 A03 VK 1069.16 * 0.0820 0.0570 1890 90 18 POL 775 PADDPSAGR A03/A11 A03 A R1150.35 0.0008 0.0002 1891 80 16 X 11 PARDVLCLR A03/A11 A03 A R 1150.360.0002 0.0002 1892 90 18 POL 385 PARVTGGVF A03/A11 A03 A F 1893 75 15EVN 83 PASTNRQSGR A03/A11 A03 A R 1894 85 17 X 68 PCALRFTSAR A03/A11 A03C R 1895 90 18 ENV 26 PDHQLDPAF A03/A11 A03 D F 1896 95 19 POL 523PFLLAQFTSA A03/A11 A03 F A 1897 95 19 POL 645 PFTQCGYPA A03/A11 A03 F A1898 100 20 ENV 244 PGYRWMCLR A03/A11 A03 G A 1.0964 0.0008 0.0005 189995 19 ENV 244 PGYRWMCLRR A03/A11 A03 G R 1.1068 0.0048 0.0001 1900 90 18POL 616 PIDWKVCQR A03/A11 A03 I R 1.0985 0.0002 0.0005 1901 100 20 ENV391 PIFFCLWVY A03/A11 A03 I Y 1.0843 0.0011 0.0002 1902 80 16 POL 496PIILGFRK A03/A11 A03 I K 1903 95 19 POL 20 PLEEELPR A03/A11 A03 L R26.0016 0.0002 −0.0002 1904 100 20 POL 438 PLHPAAMPH A03/A11 A03 L H20.0128 0.0012 0.0002 1905 95 19 ENV 174 PLLYLQAGF A03/A11 A03 L F 190695 19 ENV 174 PLLVQAGFF A03/A11 A03 L F 1907 100 20 POL 2 PLSYQHFRA03/A11 A03 L R 26.0017 −0.0002 −0.0002 1908 75 15 POL 2 PLSYQHFRKA03/A11 A03 L K 1.0161 0.0011 0.0031 1909 85 17 POL 98 PLTVNEKR A03/A11A03 L R 26.0018 0.0002 −0.0002 1910 85 17 POL 98 PLTVNEKRR A03/A11 A03 LR 1.0974 0.0008 0.0005 1911 80 16 POL 516 PMGVGLSPF A03/A11 A03 M F 191280 16 POL 516 PMGVGLSPF A03/A24 A03 M F 1913 90 18 X 20 PVGAESRGRA03/A11 A03 V R 1.0990 0.0002 0.0005 1914 85 17 POL 612 PVNRPIDWKA03/A11 A03 V K 1142.06 * 0.0310 0.1400 1915 95 19 POL 665 QAFTFSPTYA03/A11 A03 A V 20.0127 0.0030 0.0017 1916 95 19 POL 654 QAFTFSPTYKA03/A11 A03 A K 1090.10 * 0.0450 0.5400 1917 80 16 EVN 179 QAGFFLLTRA03/A11 A03 A R 1918 80 18 ENV 118 QAMQWNSTTF A03/A11 A03 A F 1919 75 15NUC 169 QSPRFFFSQSR A03/A11 A03 S R 28.0839 1920 80 16 POL 189 QSSGILSRA03/A11 A03 S R 1921 95 19 POL 539 RAFPHCLAF A03/A11 A03 A F 20.01250.0015 0.0007 1922 75 15 POL 106 RLKLIMPAR A03/A11 A03 L R 1.0975 *0.0950 0.0002 1923 75 15 POL 106 RLKLIMPARF A03/A11 A03 L F 1924 75 15 X128 RLKVFVLGGCR A03/A11 A03 L R 1925 95 19 POL 387 RLVVDFSCF A03/A11 A03L F 20.0122 0.0006 0.0002 1926 95 19 POL 376 RLWDPSQFSR A03/A11 A03 L R26.0539 * 0.2800 3.8000 1927 95 19 NUC 183 RSPRRRTPSPR A03/A11 A03 S A26.0540 −0.0007 −0.0003 1928 75 15 NUC 167 RSQSPRRR A03/A11 A03 S R 192975 15 NUC 187 RSQSPRRRR A03/A11 A03 S R 1930 95 19 NUC 188 RTPSPRRRA03/A11 A03 T R 26.0019 −0.0002 −0.0002 1931 95 19 NUC 188 RTPSPRRRRA03/A11 A03 T R 1.0971 * 0.0054 0.0005 1932 80 18 POL 829 RVHFASPLHA03/A11 A03 V H 1933 100 20 POL 357 RVTGGVFLVDK A03/A11 A03 V K1147.18 * 0.0190 0.0290 1934 90 18 X 65 SAGPCALR A03/A11 A03 A R 26.0020−0.0002 0.0020 1935 90 18 X 65 SAGPCALRF A03/A11 A03 A F 26.0152 −0.00030.0004 1936 95 19 POL 520 SAICSVVR A03/A11 A03 A R 26.0021 −0.00020.0071 1937 95 19 POL 520 SAICSVVRR A03/A11 A03 A R 1090.11 * 0.00580.2100 1938 90 18 POL 771 SALNPADDPSR A03/A11 A03 A R 26.0542 −0.0004−0.0003 1939 100 20 POL 165 SASFCGSPY A01/A03/ A03 A Y * 1940 75 15 POL759 SFPWLLGCA A03/A11 A03 F A 1941 75 15 POL 769 SFPWLLGCAA A03/A11 A03F A 1942 95 19 POL 427 SLDVSAAFYH A03/A11 A03 L H 1943 75 15 POL 565SLGHLNPNK A03/A11 A03 L K 28.0758 * 1944 100 20 ENV 348 SLLVPFVQWFA03/A11 A03 L F 1945 95 19 X 54 SLRGLPVCAF A03/A11 A03 L F 20.02590.0004 0.0002 1946 90 18 X 64 SSAGPCALR A03/A11 A03 S R 26.0153 * 0.00800.1400 1947 90 18 X 64 SSAGPCALRF A03/A11 A03 S F 26.0374 −0.0003−0.0002 1948 95 19 NUC 170 STLPETTVVR A03/A11 A03 T R 1069.21 * 0.00070.0600 1949 95 19 NUC 170 STLPETTVVRR A03/A11 A03 T R 1083.01 0.01501.4000 1950 80 16 ENV 85 STNRQSGR A03/A11 A03 T R 1951 75 15 X 104STTDLEAYF A03/A11 A03 T F 1952 75 15 X 104 STTDLEAYFK A03/A11 A03 T K1.0584 * 0.0066 2.7000 1953 95 19 POL 535 SVVRRAFPH A03/A11 A03 V H20.0131 * 0.1100 0.6100 1954 85 17 POL 727 TAELLAACF A03/A11 A03 A F1955 85 17 POL 716 TAELLAACFAR A03/A11 A03 A R 26.0544 0.0006 0.00231956 90 18 POL 747 TDNSVVLSR A03/A11 A03 D R 1957 90 18 POL 747TDNSVVLSRK A03/A11 A03 D K 20.0264 0.0006 0.0017 1958 75 15 NUC 138TFGRETVLEY A03/A11 A03 F Y 1959 95 19 POL 688 TFSPTYKAF A03/A24 A03 F F5.0064 1960 100 20 POL 370 TGGVFLVDK A03/A11 A03 G K 20.0133 0.00070.0061 1961 95 19 NUC 171 TLPETTVVR A03/A11 A03 L R 1.0969 0.0008 0.00021962 95 19 NUC 171 TLPETTVVRR A03/A11 A03 L R 1069.22 * 0.0007 0.02301963 95 19 NUC 171 TLPETTVVRRR A03/A11 A03 L R 26.0545 * 0.0005 0.01601964 100 20 POL 150 TLWKAGILY A03/A11 A03 L Y 1099.03 * 0.1300 0.00081965 100 20 POL 150 TLWKAGILYK A03/A11 A03 L K 1069.15 * 5.3000 0.36001966 100 20 POL 150 TLWKAGILYKR A03/A11 A03 L R 26.0546 0.0082 0.00951967 95 19 POL 519 TSAICSVVR A03/A11 A03 S R 5.0057 0.0005 0.0008 196895 19 POL 519 TSAICSVVAR A03/A11 A03 S R 1142.08 * 0.0018 0.0006 1969 7515 POL 756 TSFPWLLGCA A03/A11 A03 S A 1970 80 16 POL 775 TSFVYVPSAA03/A11 A03 S A 1971 75 15 X 105 TTDLEAYFK A03/A11 A03 T K 1.0215 *0.0006 0.9200 1972 75 15 EVN 278 TTSTGPCK A03/A11 A03 T K 1973 80 16 NUC175 TTVVRRRGR A03/A11 A03 T R 1.0970 0.0008 0.0005 1974 80 16 NUC 176TVVRRRGR A03/A11 A03 V R 3.0324 0.0003 0.0001 1975 80 16 NUC 176TVVRRRGRSPR A03/A11 A03 V R 28.0837 1976 100 20 POL 373 VFLVDKNPHA03/A11 A03 F H 1977 80 18 X 131 VFVLGGCRH A03/A11 A03 F H 1978 75 15 X131 VFVLGGCRHK A03/A11 A03 F K 1979 95 19 POL 637 VGLLGFAAPF A03/A11 A03G F 1980 85 17 POL 96 VGPLTVNEK A03/A11 A03 G K 20.0132 0.0007 0.00781981 85 17 POL 96 VGPLTVNEKR A03/A11 A03 G R 1982 95 19 POL 554VLGAKSVQH A03/A11 A03 L H 1983 90 18 X 133 VGGCRHK A03/A11 A03 L K26.0022 0.0150 0.0002 1984 80 16 ENV 177 VLQAGFFLLTR A03/A11 A03 L R1985 85 17 POL 752 VLSRKYTSF A03/A11 A03 L F 1986 90 18 NUC 120 VSFGWWRA03/A11 A03 S R 26.0023 * 0.0040 0.0290 1987 100 20 POL 48 VSIPWTHKA03/A11 A03 S K 26.0024 * 0.0130 0.0170 1988 100 20 POL 358 VTGGVFLVDKA03/A11 A03 T K 1069.17 * 0.0390 0.0920 1989 100 20 POL 378 VVDFSQFSRA03/A11 A03 V R 1069.19 * 0.0015 0.0750 1990 90 18 POL 553 VVLGAKSVQHA03/A11 A03 V H 1991 85 17 POL 751 VVLSRKYTSF A03/A11 A03 V F 20.02610.0004 0.0002 1992 80 16 NUC 177 VVRRRGRSPR A03/A11 A03 V R 1.10740.0027 0.0001 1993 80 16 NUC 177 VVRRRGRSPRR A03/A11 A03 V R 28.08381994 90 18 NUC 131 WFHISCLTF A03/A11 A03 F F 13.0073 * 1995 90 18 NUC131 WFHISCLTF A03/A24 A03 F F 13.0073 * 1996 85 17 NUC 28 WGMDIDPYKA03/A11 A03 G K 26.0154 −0.0003 0.0006 1997 85 17 POL 589 WGYSLNFMGYA03/A11 A03 G Y 1998 80 16 POL 770 WILRGTSFVY A03/A11 A03 I Y 1.05720.0076 0.0011 1999 95 19 NUC 125 WIRTPPAYR A03/A11 A03 I R 1.0968 0.00080.0005 2000 90 18 POL 314 WLQFRNSK A03/A11 A03 L K 26.0025 −0.00020.0005 2001 95 19 POL 425 WLSLDVSAAF A03/A11 A03 L F 2002 85 17 NUC 26WLWGMDIDPY A03/A11 A03 L Y 1.0774 * 0.0002 0.0002 2003 85 17 NUC 26WLWGMDIDPYK A03/A11 A03 L K 26.0547 0.0030 0.0013 2004 95 19 ENV 248WMCLRRFIIF A03/A11 A03 M F 20.0266 0.0004 0.0011 2005 95 19 ENV 248WMCLRRRIIF A03/A24 A03 M F 20.0266 0.0004 0.0011 2006 100 20 POL 122YLPLDKGIK A03/A11 A03 L K 1.0173 0.0001 0.0001 2007 90 18 NUC 118YLVSFGVWIR A03/A11 A03 L R 1090.13 * 0.0005 0.0002 2008 90 18 POL 538YMDDVVLGAK A03/A11 A03 M K 1090.15 * 0.0330 0.0043 2009 80 16 POL 504YSHPILGF A03/A11 A03 S F 2010 80 16 POL 493 YSHPILGFR A03/A11 A03 S R2011 80 16 POL 493 YSHPIILGFRK A03/A11 A03 S K 2012 248

TABLE XVII HBV A24 Motif With Binding Information Conservancy FrequencyProtein Position Sequence P2 C-term Peptide Filed A*2401 SEQ ID NO: 9519 X 62 AFSSAGPCAL F L 5.0118 0.0012 2013 90 18 POL 535 AFSYMDDVVL F L13.0130 0.0009 2014 80 16 ENV 108 AMQWNSTTF M F 2015 100 20 NUC 131AYRPPNAPI Y I 1090.02 * 0.0310 2016 100 20 NUC 131 AYRPPNAPIL Y L1069.24 * 0.0042 2017 90 18 NUC 117 EYLVSFGVW Y W 26.0150 2018 90 18 NUC117 EYLVSFGVWI Y I 17.0426 * 2019 80 16 ENV 182 FFLLTRILTI F I 2020 8016 ENV 181 GFFLLTRIL F L 2021 75 15 ENV 170 GFLGPLLVL F L 2022 85 17 NUC29 GMDIDPYKEF M F 26.0372 2023 85 17 ENV 65 GWSPQAQGI W I 20.0134 0.00242024 85 17 ENV 65 GWSPQAQGIL W L 20.0268 0.0003 2025 95 19 ENV 234GYRWMCLRRF Y F 1069.25 * 0.0007 2026 80 16 POL 820 HFASPLHVAW F W 2027100 20 ENV 381 IFFCLWVYI F I 5.0058 0.0087 2028 80 16 ENV 245 IFLFILLLCLF L 2029 95 19 POL 395 KFAVPNLQSL F L 5.0114 0.0020 2030 100 20 POL 121KYLPLDKGI Y I 2031 85 17 POL 745 KYTSFPWLL Y L 1069.23 * 5.3000 2032 8016 ENV 247 LFILLLCLI F I 2033 80 16 ENV 247 LFILLLCLIF F F 2034 85 17NUC 101 LWFHISCLTF W F 26.0373 2035 80 16 POL 492 LYSHPIILGF Y F2.0181 * 1.1000 2036 95 19 POL 561 NFLLSLGIHL F L 5.0115 0.0099 2037 8016 POL 758 NWILRGTSF W F 2038 95 19 POL 634 PFTQCGYPAL F L 5.0116 0.00022039 95 19 ENV 341 PFVQWFVGL F L 5.0059 0.0003 2040 80 16 POL 505PMGVGLSPF M F 2041 80 16 POL 750 PWLLGCAANW W W 2042 100 20 POL 51PWTHKVGNF W F 20.0138 * 0.0290 2043 75 15 ENV 242 RFIIFLFIL F L 2044 7515 ENV 242 RFIIFLFILL F L 2045 95 19 ENV 236 RWMCLRRFI W I 20.0135 *0.0710 2046 95 19 ENV 236 RWMCLRRFII W I 20.0269 * 1.1000 2047 100 20POL 167 SFCGSPYSW F W 20.0139 * 0.0710 2048 80 16 POL 765 SFVYVPSAL F L2049 100 20 ENV 334 SWLSLLVPF W F 20.0136 * 0.3900 2050 95 19 POL 392SWPKFAVPNL W L 20.0271 * 5.6000 2051 95 19 ENV 197 SWWTSLNFL W L20.0137 * 0.3800 2052 75 15 POL 4 SYQHFFKLL Y L 2.0042 0.0051 2053 75 15POL 4 SYQHFRKLLL Y L 2.0173 * 0.0660 2054 95 19 POL 657 TFSPTYKAF F F5.0064 0.0060 2055 95 19 POL 657 TFSPTYKAFL F L 5.0117 0.0043 2056 95 19POL 686 VFADATPTGW F W 20.0272 * 0.0180 2057 90 18 NUC 102 WFHISCLTF F F13.0073 * 0.0300 2058 95 19 ENV 345 WFVGLSPTVW F W 20.0270 * 0.0120 205995 19 ENV 237 WMCLRRFIIF M F 20.0266 0.0013 2060 48

TABLE XVIII DR SUPER MOTIF (With binding information) SEQ ID NO:Sequence Peptide DR1 DR2w2B1 DR2w2b1 DR3 DR4w4 DR4w15 DR5w11 DR5w12DR6w19 DR7 DR8w2 DR9 DRW53 2061 AANWILRGTSFVYVP 1298.07 0.0920 0.02400.0061 0.0023 0.0510 0.0250 0.0140 0.3700 0.0250 0.5800 0.2500 0.27002062 AEDLNLGNLNVSIPW 1186.01 0.0001 −0.0005 −0.0007 −0.0002 −0.00030.0170 2063 AELLAACFARSRSGA 2064 AFSYMDDVVLGAKSV 1186.02 0.0027 −0.00050.00130 2.9000 0.0006 −0.0003 −0.0005 2065 AGFFLLTRILTIPQS 1280.064.6000 0.0420 0.0190 0.0040 5.3000 0.1500 3.6000 0.0700 0.3700 3.10000.2600 1.3000 2066 AGPLEEELPRLADEG 35.0091 0.0022 2067 AKLIGTDNSVVLSRK2068 ANWILRGTSFVYVPS 2069 ARDVLCLRPVGAESR 2070 ASALYREALESPEHC 2071ASKLCLGWLWGMDID 1186.03 0.0002 −0.0005 0.0017 −0.0002 0.0013 0.0010 2072CLIFLLVLLDYQGML 2073 CLTFGRETVLEYLVS 2074 CPGYRWMCLRRFIIF 2075CPTVQASKLCLGWLW 2076 CQVGADATPTGWGLA 2077 CSVVRRAFPHCLAFS 1186.04 0.10000.1024 0.0770 0.0032 0.0016 −0.0022 0.0008 −0.0013 0.0540 0.0590 0.02501.2000 0.0460 2078 CTCIPIPSSWAFARF 2079 CWWLQFRNSKPCSDY 2080DDVVLGAKSVCHLES 2081 DEGLNRRVAEDLNLG 2082 DLNLGNLNVSIPWTH 1280.07 0.00380.0240 0.0010 2083 DVVLGAKSVQHLESL 2084 DWKVCQRIVGLLGFA 1186.05 0.0120−0.0026 0.0030 0.2500 0.0018 0.0130 2085 EIRLKVFVLGGCRHK 2086ESRLVVDFSQFSRGN 35.0096 0.0007 2.6000 2087 FFLLTRILTIPQSLD F064.01 2088FGVWIRTPPAYRPPN 2089 FIIFLFILLLCLIFL 2090 FLFILLLCLIFLLVL 2091FPWLLGCAANWILRG 2092 FRKLPVNRPIDWKVC 2093 FSWLSLLVPFVQWFV 2094FSYMDDVVLGAKSVQ 2095 FVQWFVGLSPTVWLS 1186.06 0.4700 0.0035 0.0160−0.0013 0.0130 0.0072 0.0021 0.0190 0.0690 0.0180 0.0410 0.0044 2096GAHLSLRGLPVCAFS 1186.07 0.7800 0.0042 −0.0041 0.0011 0.0025 0.00770.0150 2097 GFFLLTRILTIPQSL 1280.08 0.4300 0.0150 0.0110 3.1000 0.45002.3000 0.0780 3.5000 1.6000 0.5500 2098 GIHLNPNKTKRWGYS 2099GLPVCAFSSAGPCAL 2100 GLYFPAGGSSSGTVN 2101 GTNLSVPNPLGFFPD 2102GTSFVYVPSALNPAD 1280.09 0.3500 0.0140 0.0500 −0.0006 0.3800 0.41000.0470 −0.0001 0.0001 0.2700 0.0610 0.3400 2103 GVFLVDKNPHNTTES 2104GVGLSPFLLAQFTSA 2105 GVWIRTPPAYRPPNA 27.0280 0.3700 0.0420 7.2000 0.01203.4000 0.5700 0.4800 0.0140 −0.0004 0.2200 0.5300 0.0450 2106HGGLLGWSPQAQGIL 2107 HLPLHPAAMPHLLVG 2108 HLSLRGLPVCAFSSA 1280.10 1.30000.0028 0.0130 2109 HTALRQAILCWGELM 2110 HTLWKAGILYKRETT 2111IFLFILLLCLIFLLV 1280.11 0.0005 0.0041 0.0018 2112 IIFLFILLLCLIFLL1280.12 2113 ILGFRKIPMGVGLSP 2114 ILLLCLIFLLVLLDY F107.01 0.0026 0.00690.0320 0.0018 0.0047 2115 IRDLLDTASALYREA 2116 IRQLLWFHISCLTFG 2117IVGLLGFAAPFTQCG 1186.09 0.0200 −0.0005 −0.0007 −0.0002 0.0009 0.00672118 IWMMWYWGPSLYNIL 2119 KFAVPNLQSLTNLLS 1280.13 0.0180 0.0005 −0.00030.1300 0.0043 0.0088 −0.0003 0.0056 2120 KIPMGVGLSPFLLAQ 2121KLHLYSHPIILGFRK 2122 KQAFTFSPTYKAFLC 1298.06 0.5300 0.2400 0.1400 0.00901.1000 0.2200 0.2400 0.0024 0.0200 0.3300 0.1200 0.5400 2123KQCFRKLPVNRPIDW 1298.04 1.5000 0.0022 0.0210 −0.0006 1.2000 0.85000.0130 0.0043 0.4000 0.0580 0.0250 2124 KRRLKLIMPARFYPN 2125LAQFTSAICSVVRRA 1186.10 0.0120 0.0065 0.1500 −0.0009 0.0150 0.02800.0076 0.0091 0.0010 0.0280 0.0150 0.0880 0.0190 2126 LCLIFLLVLLDYQGMF107.02 0.0016 0.0060 0.0230 0.0017 0.0044 2127 LCQVFADATPTGWGL 1280.140.0020 0.9600 0.0013 2128 LEYLVSFGVWIRTPP 2129 LFILLLCLIFLLVLL 2130LGFFPDHQLDPAFGA 2131 LGNLNVSIPWTHKVG 2132 LGPLLVLQAGFFLLT 2133LGWLWGMDIDPYKEF 1186.12 0.0004 0.0006 0.0200 0.0280 −0.0002 0.00040.0430 2134 LHLYSHPIILGFRKI 1280.15 0.0220 0.0340 0.0400 0.0040 0.68000.1600 0.0410 0.0310 0.0002 0.0006 0.0610 0.0490 2135 LHTLWKAGILYKRET2136 LKVFVLGGCRHKLVC 2137 LLCLIFLLVLLDYQG 2138 LLDYQGMLPVCPLIP 2139LLGFAAPFTQCGYPA 2140 LLWFHISCLTFGRET 2141 LPKVLHKRTLGLSAM 2142LPLLPIFFCLWVYIZ 2143 LQSLTNLLSSNLSWL F107.03 2.5000 0.4400 0.0200−0.0013 4.8000 0.8100 0.0680 0.7500 0.0260 0.1500 0.0880 0.1100 2144LSAMSTTDLEAYFKD 2145 LSTLPETTVVRRRGR 2146 LSWLSLDVSAAFYHI 2147LTNLLSSNLSWLSLD 1186.14 0.0010 0.0083 0.0160 0.0013 0.0019 0.0200 2148LVLLDYQGMLPVCPL 1280.17 0.0034 −0.0013 0.0011 2149 LVPFVQWFVGLSPTV1186.15 0.0130 0.6900 0.0140 −0.0013 0.1500 1.4000 0.3800 0.6600 0.00180.0092 0.6600 2.5000 2.6000 2150 MQLFHLCLIISCSCP 2151 NAPILSTLPETTVVR1186.16 0.0009 0.0009 −0.0007 −0.0002 0.0005 0.1600 2152 NLNVSIPWTHKVGNF1186.17 0.0001 −0.0005 −0.0041 −0.0007 −0.0002 0.0005 0.0009 2153NLSWLSLDVSAAFYH 1186.18 0.1400 0.0003 −0.0005 1.3000 0.2900 0.00330.0022 0.0330 0.0041 0.0150 0.0620 2.4000 2154 NRPIDWKVCQRIVGL 2155PAAMPHLLVGSSGLS 2156 PDRVHFASPLHVAWR 1298.08 0.0510 0.0290 0.0008 0.00080.0054 0.0008 0.0190 0.0810 0.0035 0.2400 2157 PFLLAQFTSAICSVV F107.040.1800 0.0270 0.0042 −0.0013 0.0800 0.1200 0.0120 0.0016 0.0800 0.07700.0580 0.0590 2158 PHCLAFSYMDDVVLG 2159 PIILGFRKIPMGVGL 2160PLPIHTAELLAACFA 1280.18 0.0046 0.0490 −0.0003 2161 PPAYRPPNAPILSTL1186.20 0.0056 −0.0005 0.0038 0.0022 0.0024 0.0015 2162 PQAMQWNSTTFHQTL1298.01 0.0012 0.0300 0.1200 2163 PQSLDSWWTSLNFLG 2164 QCGYPALMPLYACIQ1186.21 0.0062 0.0018 0.0068 0.0023 0.0006 2165 QLLWFHISCLTFGRE 2166QQYVGPLTVNEKRRL 2167 QWFVGLSPTVWLSVI 2168 RDLLDTASALYREAL 1280.19 0.00010.0092 0.0770 2169 RDVLCLRPVGAESRG 2170 RFIIFLFILLLCLIF 2171RFSWLSLLVPFVQWF 1186.22 0.0430 0.0009 −0.0007 0.0002 0.0005 0.0031 2172RPGLCQVFADATPTG 2173 RQLLWFHISCLTFGR 1186.23 0.0002 0.0009 0.0140 0.00110.0061 0.0096 2174 RRAFPHCLAFSYMDD F107.05 0.0010 0.0010 −0.0009 0.00100.0017 2175 RRFIIFLFILLLCLI 2176 RRSFGVEPSGSGHID 2177 RVSWPKFAVPNLQSL2178 RWGYSLNFMGYVIGS 2179 SFGVWIRTPPAYRPP 1186.25 0.0094 0.0110 0.4300−0.0009 0.0780 0.0630 0.0260 0.0071 0.0002 0.0240 0.2500 0.0800 0.00162180 SFPWLLGCAANWILR 2181 SFVYVPSALNPADDP 2182 SGFLGPLLVLQAGFF 2183SPFLLAQFTSAICSV 1186.26 0.1200 0.0200 0.0085 −0.0013 0.0740 0.0190−0.0002 −0.0013 0.0540 0.0330 0.0014 0.0380 0.2000 2184 SSNLSWLSLDVSAAF1186.27 0.1400 0.0030 −0.0005 1.5000 0.2700 0.0046 0.0180 0.1000 0.00390.0460 0.0110 6.2000 2185 SVELLSFLPSDFFPS 2186 SVRFSWLSLLVPFVQ 1280.200.9000 0.0099 0.0037 2187 SVVLSRKYTSFPWLL 27.0282 0.0005 0.0057 0.2100−0.0016 0.5300 0.0130 2188 TNFLLSLGIHLINPK 1298.03 3.5000 0.0410 0.12000.0220 0.0360 0.0053 0.0160 0.2200 0.0032 0.3800 2189 TNLLSSNLSWLSLDV1186.26 0.0016 −0.0005 0.1300 0.0006 0.0019 0.0410 2190 TRILTIPQSLDSWWT2191 TSFVYVPSALNPADD 2192 TSGFLGPLLVLQAGF 2193 VAPLPIHTAELLAAC 2194VCAFSSAGPCALRFT 1186.29 0.2100 0.2600 0.0023 0.0003 0.0200 0.0150 2195VELLSFLPSDFFPSI 2196 VGLLGFAAPFTQCGY 1280.21 0.0470 0.3100 0.0008−0.0014 −0.0004 −0.0001 0.0014 0.5700 2197 VGNFTGLYSSTVPVF 1298.021.7000 0.0100 0.0016 0.0140 0.1700 0.0035 0.0580 0.5600 0.0044 0.31002198 VLCLRPVGAESRGRP 2199 VQWFVGLSPTVWLSV 2200 WASVRFSWLSLLVPF 2201WLSLDVSAAFYHIPL 2202 WLSLLVPFVQWFVGL 2203 WMCLRRFIIFLFILL 2204WPKFAVPNLQSLTNL 1186.30 0.0007 0.0013 0.0023 0.0002 0.0008 0.0180 2205YPALMPLYACIQSKQ 1298.05 0.2400 0.0014 0.0011 145

TABLE XIX HBV DR3 MOTIF PEPTIDES WITH BINDING DATA Binding Total CoreCore Pro- Posi- Core Data Conservancy Total Convervancy Freq. tein tionCore Sequence SEQ ID NO: Sequence SEQ ID NO: Peptide Filed DR3 Motif90.00 18 90.00 18 POL 535 YMDDVVLGA 2228 AFSYMDDVVLGAKSV 2206 1186.020.0130 DR3 55.00 11 95.00 19 POL 655 FSPTYKAFL 2229 AFTFSPTYKAFLCKQ 220735.0099 0.0035 DR3 65.00 13 90.00 18 POL 18 LEEELPRLA 2230AGPLEEELPRLADEG 2208 35.0091 0.0022 DR3 65.00 13 80.00 16 POL 731IGTDNSVVL 2231 AKLIGTDNSVVLSRK 2209 DR3 85.00 17 85.00 17 NUC 34LYREALESP 2232 ASALYREALESPEHC 2210 DR3 70.00 14 75.00 15 NUC 136FGRETVLEY 2233 CLTFGRETVLEYLVS 2211 DR3 90.00 18 90.00 18 x 48 AHLSLRGLP2234 DHGAHLSLRGLPVCA 2212 DR3 85.00 17 90.00 18 POL 737 VVLSRKYTS 2235DNSVVLSRKYTSFPW 2213 DR3 45.00 9 100.00 20 POL 374 LVVDFSQFS 2236ESRLVVDFSQFSRGN 2214 35.0096 * 2.6000 DR3 5.00 1 75.00 1 ENV 172AVLDPRVRG 2237 FHQAVLDPRVRGLYL 2215 DR3 90.00 18 95.00 19 ENV 256VLLDYQGML 2238 FLLVLLDYQGMLPVC 2216 35.009 0.0170 DR3 55.00 11 100.00 20POL 360 FLVDKNPHN 2239 GGVFLVDKNPHNTTE 2217 35.0095 0.0790 DR3 95.00 1995.00 19 POL 683 VFADATPTG 2240 LCQVFADATPTGWGL 2218 1280.14 0.0000 DR335.00 7 95.00 19 X 18 VGAESRGRP 2241 LRPVGAESRGRPVSG 2219 35.0101−0.0017 DR3 55.00 11 95.00 19 POL 412 LSLDVSAAF 2242 LSWLSLDVSAAFYHI2220 DR3 45.00 9 85.00 17 NUC 27 MDIDPYKEF 2243 LWGMDIDPYKEFGAS 2221 DR385.00 17 100.00 20 POL 34 VAEDLNLGN 2244 NRRVAEDLNLGNLNV 2222 35.00920.1400 DR3 100.00 20 100.00 20 POL 47 IPWTHKVGN 2245 NVSIPWTHKVGNFTG2223 DR3 45.00 9 95.00 19 ENV 10 FFPDHQLDP 2246 PLGFFPDHQLDPAFG 2224 DR330.00 6 75.00 15 POL 241 FGVEPSGSG 2247 RRSFGVEPSGSGHID 2225 DR3 100.0020 100.00 20 POL 120 LPLDKGIKP 2248 TKYLPLDKGIKPYYP 2226 35.0094 −0.0017DR3 60.00 12 85.00 17 POL 96 LTVNEKRRL 2249 VGPLTVNEKRRLKLI 222735.0093 * 2.2000 DR3 22 22

TABLE XX Population coverage with combined HLA Supertypes PHENOTYPICFREQUENCY HLA-SUPERTYPES Caucasian N.A. Black Japanese Chinese HispanicAverage A2, A3, B7 83.0 86.1 87.5 88.4 86.3 86.2 A2, A3, B7, A24, 99.598.1 100.0 99.5 99.4 99.3 B44, A1 A2, A3, B7, A24, 99.9 99.6 100.0 99.899.9 99.8 B44, A1, B27, B62, B58

TABLE XXI HBV ANALOGS A2 A3 B7 1° A1 Super Super A24 Super Anchor AASequence Fixed Nomen. Motif Motif Motif Motif Motif Fixer Analog SEQ IDNO: ALFKDWEEL 2250 9 ALMPLYACV L2.IV9 N Y N N N 1 A 2251 ALMPLYASI 22529 ALMPLYAXI N Y N N N A 2253 10 ALPSDFFPSV N Y N N N No A 2254ALPSDFFPSV-NH2 2255 ALSLIVNLL 2256 9 AMTFSPTYK N N Y N N A 2257ATVELLSFLPSDFFPSV-NH2 2258 10 CILLLCLIFL N Y N N N No A 2259 11CILLLCLIFLL N Y N N N No A 2260 9 DPFRGRLGL N N N N Y A 2261 9 DPSRGRLGIN N N N Y A 2262 ELLSFLPSDFFPSV-NH2 2263 10 FAPSDFFPSV LA2.V10 N Y N N NRev A 2264 10 FILLLXLIFL N Y N N N A 2265 10 FLASDFFPSV N Y N N N No A2266 10 FLGLSPTVWV VL2.LV1 N Y N N N 1 A 2267 10 FLKSDFFPSV N Y N N N NoA 2268 10 FLLAQFTSAV L2.IV10 N Y N N N 1 A 2269 9 FLLAQFTSV L2.AV9 N Y NN N 1 A 2270 9 FLLPIFFCL N Y N N N No A 2271 9 FLLSLGIHV L2.LV9 N Y N NN 1 A 2272 9 FLLTRILTV L2.IV9 N Y N N N 1 A 2273 9 FLLTRILYI N Y N N N A2274 9 FLLTYILTI N Y N N N A 2275 10 FLMSDYFPSV N Y N N N No A 2276 9FLMSYFPSV N Y N N N No A 2277 10 FLPADFFPSI L2.SA4 N Y N N N Rev A 227810 FLPADFFPSV N Y N N N No A 2279 10 FLPDDFFPSA L2.SD4 N Y N N N Rev A2280 10 FLPDDFFPSV N Y N N N No A 2281 10 FLPNDFFPSA L2.SN4 N Y N N NRev A 2282 10 FLPNDFFPSV N Y N N N No A 2283 10 FLPS(X)YFPSV N N N N N A2284 10 FLPSAFFPSV N Y N N N No A 2285 10 FLPSD(X)FPSV N N N N N A 228610 FLPSDAFPSV N Y N N N No A 2287 10 FLPSDFAPSV N Y N N N No A 2288FLPSDFF-NH2 2289 10 FLPSDFFASV N Y N N N No A 2290 10 FLPSDFFKSV N Y N NN No A 2291 8 FLPSDFFP N N N N N A 2292 FLPSDFFP-NH2 2293 10 FLPSDFFPAVN Y N N N No A 2294 10 FLPSDFFPKV N Y N N N No A 2295 9 FLPSDFFPS N N NN N A 2296 FLPSDFFPS-NH2 2297 10 FLPSDFFPSA LV.VA10 N Y N N N Rev A 229810 FLPSDFFPSI LV.VI10 N Y N N N Rev A 2299 FLPSDFFPSV(CONH2) 2300FLPSDFFPSV-NH2 2301 11 FLPSDFFPSVR N N Y N N A 2302 FLPSDFFPSVR-NH2 230312 FLPSDFFPSVRD N N N N N A 2304 10 FLPSDFYPSV N Y N N N No A 2305 11FLPSDLLPSVR N N Y N N A 2306 10 FLPSDYFPSV N Y N N N No A 2307 10FLPSEFFPSV N Y N N N No A 2308 9 FLPSYFPSA L2.FY5. N Y N N N Rev3 A 23099 FLPSYFPSV L2.FY5. N Y N N N 3 A 2310 10 FLPSZFFPSV N Y N N N No A 231110 FLPSZFFPSV N Y N N N No A 2312 10 FLPVDFFPSI L2.SV4. N Y N N N Rev A2313 10 FLPVDFFPSV N Y N N N No A 2314 FLSKQYLNL 2315 9 FLYTRILTI N Y NN N A 2316 8 FMFSPTYK N N Y N N A 2317 10 FMLLLCLIFL IM2.L10 N Y N N N 1A 2318 10 FMPSDFFPSV LM2.V1 N Y N N N 1 A 2319 8 FPAAMPHL N N N N Y A2320 9 FPAAMPHLL N N N N Y A 2321 10 FPAAMPHLLV N N N N Y A 2322 9FPALMPLYA N N N N Y A 2323 10 FPARVTGGVF N N N N Y A 2324 10 FPCALRFTSAN N N N Y A 2325 9 FPFCLAFSY N N N N Y A 2326 10 FPFCLAFSYM N N N N Y A2327 9 FPHCLAFAL N N N N Y A 2328 9 FPHCLAFAY N N N N Y A 2329 9FPHCLAFSA N N N N Y A 2330 9 FPHCLAFSI N N N N Y A 2331 9 FPHCLAFSL N NN N Y A 2332 10 FPHCLAFSYI N N N N Y A 2333 10 FPHXLAFSYM N N N N Y A2334 9 FPIPSSWAF N N N N Y A 2335 9 FPSRGRLGL N N N N Y A 2336 9FPVCAFSSA N N N N Y A 2337 9 FPVCLAFSY N N N N Y A 2338 10 FQPSDYFPSV NN N N Y Rev A 2339 8 FVFSPTYK N N Y N N A 2340 9 FVLGGXRHK N N Y N N A2341 9 GLCQVGADV L2.AV9 N Y N N N 1 A 2342 9 GLLGWSPQV L2.AV9 N Y N N N1 A 2343 9 GLWIRTIPPV VL2.AV9 N Y N N N 1 A 2344 9 GLXQFADA N Y N N N A2345 10 GMDNSVVLSR N N Y N N A 2346 11 GMDNSVVLSRK N N Y N N A 2347 10GPCALRFTSI N N N N Y A 2348 10 GPFALRFTSA N N N N Y A 2349 10 GPXALRFTSAN N N N Y A 2350 10 GTFNSVVLSR N N Y N N A 2351 11 GTFNSVVLSRK N N Y N NA 2352 10 GVDNSVVLSR N N Y N N A 2353 11 GVDNSVVLSRK N N Y N N A 2354 10GYRWMXLRRF N N N Y N A 2355 9 HISXLTFGR N N Y N N A 2356 10 HMLWKAGILY YN Y N N A 2357 11 HMLWKAGILYK N N Y N N A 2358 8 HPAAMPHI N N N N Y A2359 9 HPAAMPHLI N N N N Y A 2360 10 HPAAMPHLLI N N N N Y A 2361 8HPFAMPHL N N N N Y A 2362 9 HPFAMPHLL N N N N Y A 2363 10 HPFAMPHLLV N NN N Y A 2364 10 HTLWKAGILK N N Y N N A 2365 10 HTLWKAGILR N N Y N N A2366 10 HVLWKAGILY N N Y N N A 2367 11 HVLWKAGILYK N N Y N N A 2368IIKKSEQFV 2369 9 ILGLLGFAV VL2.AV9 N Y N N N 1 A 2370 10 ILLLCLIFLVL2.LV10 N Y N N N 1 A 2371 9 ILLLXLIFL N Y N N N A 2372 10 ILLLXLIFLL NY N N N A 2373 9 IPFPSSWAF N N N N Y A 2374 9 IPILSSWAF N N N N Y A 23759 IPIPMSWAF N N N N Y A 2376 9 IPIPSSWAI N N N N Y A 2377 9 IPITSSWAF NN N N Y A 2378 KIKESFRKL N N N N Y A 2379 9 KLFLYSHPI N Y N N N No A2380 9 KLHLYSHPV L2.IV9 N Y N N N 1 A 2381 9 KVGNFTGLK N N Y N N A 23829 KVGNFTGLR N N Y N N A 2383 9 LLAQFTSAV L2.IV9 N Y N N N 1 A 2384 10LLFYQGMLPV N Y N N N No A 2385 LLGSAANWI 2386 10 LLGXAANWIL N Y N N N A2387 9 LLLXLIFLL N Y N N N A 2388 10 LLLXLIFLLV N Y N N N A 2389 10LLLYQGMLPV N Y N N N No A 2390 9 LLPFVQWFV VL2.V9 N Y N N N 1 A 2391 10LLPIFFXLWV N Y N N N A 2392 LLSFLPSDFFPSV-NH2 2393 9 LLSSNLSWV L2.LV9 NY N N N 1 A 2394 10 LLVLQAGFFV L2.LV10 N Y N N N 1 A 2395 9 LLXLIFLLV NY N N N A 2396 10 LMLLDYQGMV VM2.LV N Y N N N 1 A 2397 10 LMLQAGFFLVVM2.LV N Y N N N 1 A 2398 9 LMPFVQWFV VM2.V9 N Y N N N 1 A 2399 9LPFCAFSSA N N N N Y A 2400 8 LPIFFCLI N N N N Y A 2401 9 LPIFFCLWI N N NN Y A 2402 9 LPIHTAELI N N N N Y A 2403 11 LPIHTAELLAI N N N N Y A 2404LPSDFFPSV-NH2 2405 9 LPVCAFSSI N N N N Y A 2406 9 LPVXAFSSA N N N N Y A2407 12 LSFLPSDFFPSV N N N N N A 2408 LSFLPSDFFPSV-NH2 2409 10MMWYWGPSLK N N Y N N A 2410 10 MMWYWGPSLR N N Y N N A 2411 9 MMWYWGPSVM2.LV9 N Y N N N 1 A 2412 8 MPLSYCHI N N N N Y A 2413 9 NLGNLNVSV L2.IV9N Y N N Y 1 A 2414 NLNNLNVSI 2415 9 NMGLKYRQL N Y N Y N No A 2416 11NPLGFFPDHQI N N N N Y A 2417 9 PLLPIFFCV L2.LV9 N Y N N N 1 A 2418 9PLLPIFFXL N Y N N N A 2419 8 PSDFFPSV N N N N N A 2420 PSDFFPSV-NH2 242110 QLLWFHSXL N Y N N N A 2422 10 QMFTFSPTYK N N Y N N A 2423 10QVFTFSPTYK N N Y N N A 2424 RIPRTPRSV 2425 9 RLSWPKFAV VL2.V9 N Y N N N1 A 2426 9 RLTGGVFLV VL2.V9 N Y N N N 1 A 2427 9 RMLTIPQSV IM2.LV9 N Y NN N 1 A 2428 9 RMTGGVFLV VM2.V9 N Y N N N 1 A 2429 9 RMYLHTLWK N N Y N NA 2430 9 RVYLHTLWK N N Y N N A 2431 9 SAIXSVVRR N N Y N N A 2432 11SFLPSDFFPSV N N N N N A 2433 SFLPSDFFPV-NH2 2434 9 SLDSWWTSV L2.LV9 N YN N N 1 A 2435 SLNFLGGTTV(NH2) L2.LV9 N Y N N N 1 A 2436 9 SMICSVVRR N NY N Y A 2437 10 SMLPETTVVR N N Y N N A 2438 11 SMLPETTVVRR N N Y N N A2439 10 SMLSPFLPLY IM2.LV1 N Y N N N 1 A 2440 8 SPFLLAQI N N N N Y A2441 11 STLPETYVVRR N N Y N N A 2442 9 SVICSVVRR N N Y N N A 2443 10SVLPETTVVR N N Y N N A 2444 11 SVLPETTVVRR N N Y N N A 2445 9 SVNRPIDWKN N Y N N A 2446 9 SVVRRAFPK N N Y N N A 2447 9 SVVRRAFPR N N Y N N A2448 9 TLWKAGILK N N Y N N A 2449 9 TLWKAGILR N N Y N N A 2450 10TMPETTVVRR N N Y N N A 2451 9 TMWKAGILY Y N Y N N A 2452 10 TMWKAGILYK NN Y N N A 2453 10 TPARVTGGVI N N N N Y A 2454 10 TPFRVTGGVF N N N N Y A2455 10 TSAIXSVVRR N N Y N N A 2456 TVELLSFLPSDFFPSV-NH2 2457 10TVPETTVVRR N N Y N N A 2458 9 TVWKAGILY N N Y N N A 2459 10 TVWKAGILYK NN Y N N A 2460 VELLSFLPSDFFPSV-NH2 2461 VLEYLVSFGV(NH2) 2462 VLGGSRHKL2463 9 VLLDYQGMV L2.LV9 N Y N N N 1 A 2464 9 VLQAGFFLV L2.LV9 N Y N N N1 A 2465 10 VMGGVFLVDK N N Y N N A 2466 10 VPFVQWFVGI N N N N Y A 2467 8VPSALNPI N N N N Y A 2468 9 VVFFSQFSR N N Y N N A 2469 10 VVGGVFLVDK N NY N N A 2470 9 WLLRGTSFV IL2.V9 N Y N N N 1 A 2471 10 YLFTLWKAGI N Y N NN No A 2472 10 YLHTLWKAGV L2.IV10 N Y N N N 1 A 2473 10 YLLTLWKAGI N Y NN N No A 2474 9 YLLTRILTI N Y N N N A 2475 9 YLPSALNPV VL2.AV9 N Y N N N1 A 2476 10 YLPSDFFPSV N Y N N N No A 2477 9 YMDDVVLGV M2.AV9 N Y N N N1 A 2478 9 YMFDVVLGA N Y N N N No A 2479 10 YMFDVVLGAK N N Y N N A 248010 YNMGLKFRQL N N N N N A 2481 8 YPALMPLI N N N N N A 2482 9 YPALMPLYI NN N N Y A 2483 9 YPFLMPLYA N N N N Y A 2484 12 YSFLPSDFFPSV N N N N N A2485 237

TABLE XXII Discreet substitutions improve the B7 supertype bindingcapacity and degeneracy of peptide ligands. Binding (IC₅₀ nM) Source 1 23 4 5 6 7 8 9 B*0701 B*3501 B*5101 B*5301 B*5401 x-rxn SEQ ID NO: HBVENV I P I P S S A F 42 2.6 2.3 12 2970 4 2505 313 F P I P S S A F 24 1.2305 1.7 105 5 2506 I P I P S S A I 31 54 15 24 7.7 5 2507 HBV POL 541 FP H C L A F S Y — 14 83 17 503 3 2508 F P H C L A F A L 25 2.7 28 5.0 245 2509 F P H C L A F S L 74 2.4 4.5 15 7.7 5 2510 F P F C L A F S Y —6.5 27 4.8 5.1 4 2511 F P H C L A F S I 675 29 6.3 3.8 1.0 4 2512 F P HC L A F S A 3667 6.5 250 137 0.6 4 2513 HCV Core 168 L P G C S F S I F28 90 100 114 6897 4 2514 F P G C S F S I F 19 1.6 132 3.2 67 5 2515MAGE2 170 V P I S H L Y I L 22 171 96 238 3175 4 2516 F P I S H L Y I L16 7.3 6.4 7.0 28 5 2517 MAGE3 196 P K A G L L I I 940 5039 393 90 248 32518 F P K A G L L I I 162 1303 5.8 60 150 4 2519 P F A G L L I I 2291.0 0.9 2.3 0.27 5 2520

TABLE XXIII Sets of preferred epitopes restricted by class I and classII molecules can be selected for inclusion in an HBV-specific vaccine.Table XXIII lists as a matter of example one such set of epitopes. SEQrestric- ID Peptide Sequence Protein tion NO: A) Class I restrictedepitopes 924.07 FLPSDFFPSV core 18 A2 2521 777.03 FLLTRILTI env 183 A22522 927.15 ALMPLYACI pol 642 A2 2523 1013.01 WLSLLVPFV env 335 A2 25241090.14 YMDDVVLGA pol 538 A2/A1 2525 1168.02 GLSRYVARL pol 455 A2 2526927.11 FLLSLGIHL pol 562 A2 2527 1069.10 LLPIFFCLWV env 378 A2 25281069.06 LLVPFVQWFV env 338 A2 2529 1147.16 HTLWKAGILYK pol 149 A3/A12530 1083.01 STLPETTVVRR core 141 A3 2531 1069.16 NVSIPWTHK pol 47 A32532 1069.20 LVVDFSQFSR pol 388 A3 2533 1090.10 QAFTFSPTYK pol 665 A32534 1090.11 SAICSVVRR pol 531 A3 2535 1142.05 KVGNFTGLY pol 629 A3/A12536 1147.05 FPHCLAFSYM pol 530 B7 2537 988.05 LPSDFFPSV core 19 B7 25381145.04 IPIPSSWAF env 313 B7 2539 1147.02 HPAAMPHLL pol 429 B7 254026.0570 YPALMPLYACI pol 640 B7 2541 1147.04 TPARVTGGVF pol 354 B7 25421.0519 DLLDTASALY core 419 A1 2543 2.0239 LSLDVSAAFY pol 1000 A1 25441039.06 WMMWYWGPSLY env 359 A1 2545 20.0269 RWMCLRRFII env 236 A24 254620.0136 SWLSLLVPF env 334 A24 2547 20.0137 SWWTSLNFL env 197 A24 254813.0129 EYLVSFGVWI core 117 A24 2549 1090.02 AYRPPNAPI core 131 A24 255013.0073 WFHISCLTF core 102 A24 2551 20.0271 SWPKFAVPNL pol 392 A24 25521069.23 KYTSFPWLL pol 745 A24 2553 2.0181 LYSHPIILGF pol 492 A24 2554 B)Glass II restricted epitopes F107.03 LQSLTNLLSSNLSWL pol 412 DR 2555super- motif 1298.06 KQAFTFSPTYKAFLC pol 664 2556 1280.06AGFFLLTRILTIPQS env 180 2557 1280.09 GTSFVYVPSALNPAD pol 774 2558 CF-08VSFGVWIRTPPAYRPP- core 120 2559 NAPI  27.0281 RHYLHTLWKAGILYK pol 1452560 1186.15 LVPFVQWFVGLSPTV env 339 2561 1280.15 LHLYSHPIILGFRKI pol501 2562 F107.04 PFLLAQFTSAICSVV pol 523 2563 1298.04 KQCFRKLPVNRPIDWpol 618 2564 1298.07 AANWILRGTSFVYVP pol 767 2565 857.02PHHTALRQAILCWGEL- core 50 2566 MTLA 1280.14 LCQVFADATPTGWGL pol 694 DR32567 motif 35.0096 ESRLVVDFSQFSRGN pol 385 2568 35.0093 VGPLTVNEKRRLKLIpol 96 2569 1186.27 SSNLSWLSLDVSAAF pol 420 2570 1186.18 NLSWLSLDVSAAFYHpol 442 2571

1. An isolated peptide consisting of the oligopeptide LWFHISCLTF (SEQ IDNO:879).
 2. A composition comprising the peptide of claim 1 and acarrier.
 3. A composition comprising the peptide of claim 1 and apharmaceutically acceptable carrier.
 4. A composition comprising thepeptide of claim 1 and a liposome.
 5. A composition comprising thepeptide of claim 1, wherein the peptide is admixed with at least oneother peptide comprising a helper T lymphocyte (HTL) epitope or isjoined to at least one other peptide consisting of an HTL epitope.